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  • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
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  • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
  • C04B35/622—Forming processes; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
  • C04B35/626—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B
  • C04B35/63—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B using additives specially adapted for forming the products, e.g.. binder binders
  • C04B35/632—Organic additives
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
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  • A61P11/00—Drugs for disorders of the respiratory system
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  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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  • A61P37/00—Drugs for immunological or allergic disorders
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/06—Antianaemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
  • C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2318/00—Antibody mimetics or scaffolds
  • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes

Definitions

• the present invention relates to hinge core mimetibodies, specified portions and variants specific for bologically active proteins, fragment or ligands, hinge core mimetibody encoding and complementary nucleic acids, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices.
• Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formuiation and chemical modification. Such modifications can potentially enhance the therapeutic utility of therapeutic proteins, such as by increaseing half lives (e.g., by blocking their exposure to proteolytic enzymes), enhancing biological activity, or reducing unwanted side effects.
• One such modification is the use of immunoglobulin fragments fused to receptor proteins, such as enteracept.
• Therapeutic proteins have also been constructed using the Fc domain to attempt to provide a longer half-life or to incorporate functions such as Fc receptor binding, protein A binding, and complement fixation. Accordingly, there is a need to provide improved and/or modified versions of therapeutic proteins, which overcome one more of these and other problems known in the art.
• the present invention provides isolated human hinge core mimetibodies, including modified immunoglobulins, cleavage products and other specified portions and variants thereof, as well as hinge core mimetibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and/or enabled herein, in combination with what is known in the art.
• the present invention also provides at least one hinge core mimetibody or specified portion or variant as described herein and/or as known in the art.
• the hinge core mimetibody can optionally comprise at least one CH3 region directly linked with at least one CH2 region directly linked with at least one portion of a truncated hinge region or fragment thereof (H) directly linked with at an optional linker sequence (L), directly linked to at least one therapeutic peptide (P), optionally further directly linked with at least a portion of at least one variable antibody sequence (V).
• a pair of a IgG CH3-CH2-partial hinge(H) linker (L)-therapeutic peptide (P) with an optional N-terminal variable sequence the pair optionally linked by association or covalent linkage, such as, but not limited to, at least one Cys-Cys disulfide bond or at least one CH4 or other immunglobulin sequence.
• a hinge core mimetibody comprises formula (I): ((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portion of an N-terminus of an immunoglobulin variable region, P is at least one bioactive peptide, L is at least one linker polypeptide H is at least one portion of at least one immunoglobulin hinge region, CH2 is at least a portion of an immunoglobulin CH2 constant region, CH3 is at least a portion of an immunoglobulin CH3 constant region, m, n, o, p, q, r and s are independently an integer between 0, 1 or 2 and 10, mimicing different types of immunoglobulin molecules, e.g., but not limited to IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, and the like, or any subclass thereof, or any combination thereof.
• a hinge core mimetibody of the present invention mimics at least a portion of an antibody or immnuoglobulin structure or function with its inherent properties and functions, while providing a therapeutic peptide and its inherent or acquired in vitro, in vivo or in situ properties or activities.
• the various portions ofthe antibody and therapeutic peptide portions of at least one hinge core mimetibody ofthe present invention can vary as described herein in combination with what is known in the art.
• At least one hinge core mimetibody or specified portion or variant ofthe invention mimics the binding ofthe P portion ofthe mimetibody to at least one ligand, or has at least one biological activity of, at least one protein, subunit, fragment, portion or any combination thereof.
• the present invention also provides at least one isolated hinge core mimetibody or specified portion or variant as described herein and/or as known in the art, wherein the hinge core mimetibody or specified portion or variant has at least one activity, such as, but not limited to known biological activities of at least one bioactive peptide or polypeptide corresponding to the P portion of Formula I.
• a hinge core mimetibody can thus be screened for a corresponding activity according to known methods, such as at least one neutralizing activity towards a protein or fragment thereof.
• the present invention provides at least one isolated hinge core mimetibody, comprising at least one P(n) region comprising at least a bilogically active portion of at least one of SEQ ID NOS: 1-979, or optionally with one or more substitutions, deletions or insertions as described herein and/or as known in the art.
• the present invention provides at least one isolated hinge core mimetibody, wherein the hinge core mimetibody specifically binds at least one epitope comprising at least 1-3, to the entire amino acid sequence , of at least one ligand or binding region which ligand binds to at least a portion of at least one of SEQ ID NOS: 1-979, or optionally with one or more substitutions, deletions or insertions as described herein or as known in the art.
• the at least one hinge core mimetibody can optionally further comprise at least one characteristic selected from (i) bind at least one protein with an affinity of at least IO "9 M, at least 10 "10 M, at least 10 "11 M, or at least IO "12 M; and/or (ii) substantially neutralize at least one activity of at least one protein or portion thereof.
• the present invention provides, in one aspect, isolated nucleic acid molecules comprising, complementary, having significant identity or hybridizing to, a polynucleotide encoding specific mimetibodies or specified portions or variants thereof, comprising at least one specified sequence, domain, portion or variant thereof.
• the present invention further provides recombinant vectors comprising at least one of said isolated hinge core mimetibody nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such hinge core mimetibody nucleic acids, vectors and/or host cells.
• an isolated nucleic acid encoding at least one isolated hinge core mimetibody; an isolated nucleic acid vector comprising the isolated nucleic acid, and/or a prokaryotic or eukaryotic host cell comprising the isolated nucleic acid.
• the host cell can optionally be at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cell thereof.
• a method for producing at least one hinge core mimetibody comprising translating the hinge core mimetibody encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the hinge core mimetibody is expressed in detectable or recoverable amounts.
• the present invention also provides at least one composition
• a composition comprising (a) an isolated hinge core mimetibody or specified portion or variant encoding nucleic acid and/or hinge core mimetibody as described herein; and (b) a suitable carrier or diluent.
• the carrier or diluent can optionally be pharmaceutically acceptable, according to known methods.
• the composition can optionally further comprise at least one further compound, protein or composition.
• a composition comprising at least one isolated hinge core mimetibody and at least one pharmaceutically acceptable carrier or diluent.
• the composition can optionally further comprise an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, an anti-infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplactic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a nutritional drug, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythrop
• the present invention further provides at least one anti-idiotype antibody to at least one hinge core mimetibody ofthe present invention.
• the anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complimetarity determing region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into a hinge core mimetibody ofthe present invention.
• CDR complimetarity determing region
• a hinge core mimetibody ofthe invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, and the like.
• the present invention further provides an anti-idiotype antibody or fragment that specifically binds at least one hinge core mimetibody ofthe present invention.
• the present invention provides, in one aspect, isolated nucleic acid molecules comprising, complementary, or hybridizing to, a polynucleotide encoding at least one hinge core mimetibody anti-idiotype antibody, comprising at least one specified sequence, domain, portion or variant thereof.
• the present invention further provides recombinant vectors comprising said hinge core mimetibody anti-idiotype antibody encoding nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such anti-idiotype antiobody nucleic acids, vectors and/or host cells.
• the present invention also provides at least one method for expressing at least one hinge core mimetibody or specified portion or variant, or hinge core mimetibody anti-idiotype antibody, in a host cell, comprising culturing a host cell as described herein and/or as known in the art under conditions wherein at least one hinge core mimetibody or specified portion or variant, or anti-idiotype antibody is expressed in detectable and/or recoverable amounts
• the present invention further provides at least one hinge core mimetibody, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of at least one of a bone and joint disorder, cardiovascular disoder, a dental or oral disorder, a dermatologic disorder, an ear, nose or throat disorder, an endocrine or metabolic disorder, a gastrointestinal disorder, a gynecologic disorder, a hepatic or biliary disorder, a an obstetric disorder, a hematologic disorder, an immunologic or allergic disorder,
• the present invention further provides at least one hinge core mimetibody, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of, at least one immune, cardiovascular, infectious, malignant, and/or neurologic disease in a cell, tissue, organ, animal or patient and/or, as needed in many different conditions, such as but not limited to, prior to, subsequent to, or during a related disease or treatment condition, as l ⁇ iown in the art and/or as described herein.
• the present invention also provides at least one composition, device and/or method of delivery of a therapeutically or prophylactically effective amount of at least one hinge core mimetibody or specified portion or variant, according to the present invention.
• the present invention also provides at least one composition
• at least one composition comprising (a) an isolated hinge core mimetibody encoding nucleic acid and/or hinge core mimetibody as described herein; and (b) a suitable carrier or diluent.
• the carrier or diluent can optionally be pharmaceutically acceptable, according to known carriers or diluents.
• the composition can optionally further comprise at least one further compound, protein or composition.
• the present invention further provides at least one hinge core mimetibody method or composition, for administering a therapeutically effective amount to modulate or treat at least one protein related condition in a cell, tissue, organ, animal or patient and/or, prior to, subsequent to, or during a related condition, as known in the art and/or as described herein.
• the present invention also provides at least one composition, device and or rretho ⁇ iof delivery of a therapeutically or prophylactically effective amount of at least one hinge core mimetibody, according to the present invention.
• the present invention further provides at least one hinge core mimetibody method or composition, for diagnosing at least one protein related condition in a cell, tissue, organ, animal or patient and/or, prior to, subsequent to, or during a related condition, as known in the art and/or as described herein.
• the present invention also provides at least one composition, device and/or method of delivery for diagnosing of at least one hinge core mimetibody, according to the present ' invention.
• Also provided is a method for diagnosing or treating a disease condition in a cell, tissue, organ or animal comprising (a) contacting or administering a composition comprising an effective amount of at least one isolated hinge core mimetibody ofthe invention with, or to, the cell, tissue, organ or animal.
• the method can optionally further comprise using an effective amount of 0.001-50 mg/kilogram ofthe cells, tissue, organ or animal.
• the method can optionally further comprise using the contacting or the administrating by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
• parenteral subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary,
• the method can optionally further comprise administering, prior, concurrently or after the (a) contacting or administering, at least one composition comprising an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, an anti-infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplactic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a nutritional drug, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non- steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial, an antip
• a medical device comprising at least one isolated hinge core mimetibody ofthe invention, wherein the device is suitable to contacting or administerting the at least one hinge core mimetibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
• parenteral subcutaneous, intramuscular, intravenous, intrarticular, intra
• an article of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or a lyophilized form of at least one isolated hinge core mimetibody ofthe present invention.
• the article of manufacture can optionally comprise having the container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or
• a method for producing at least one isolated hinge core mimetibody of the present invention comprising providing a host cell or transgenic animal or transgenic plant or plant cell capable of expressing in recoverable amounts the hinge core mimetibody. Further provided in the present invention is at least one hinge core mimetibody produced by the above method. The present invention further provides any invention described herein. DESCRIPTION OF THE INVENTION The present invention provides isolated, recombinant and/or synthetic mimetibodies or specified portions or variants, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one hinge core mimetibody.
• Such mimetibodies or specified portions or variants ofthe present invention comprise specific hinge core mimetibody sequences, domains, fragments and specified variants thereof.
• the present invention also provides methods of making and using said nucleic acids and mimetibodies or specified portions or variants, including therapeutic compositions, methods and devices.
• the present invention also provides at least one isolated hinge core mimetibody or specified portion or variant as described herein and/or as known in the art.
• the hinge core mimetibody can optionally comprise at least one CH3 region directly linked with at least one CH2 region directly linked with at least one hinge region or fragment thereof (H) directly linked with at least one optional linker sequence (L), directly linked to at least one therapeutic peptide (P), optionally further directly linked with at least a portion of at least one variable (V) antibody sequence.
• a hinge core mimetibody comprises formula (I):
• V is at least one portion of an N-terminus of an immunoglobulin variable region
• P is at least one bioactive peptide
• L is polypeptide that provides structural flexablity by allowing the mimietibody to have alternative orientations and binding properties
• H is at least a portion of an immunoglobulin variable hinge region
• CH2 is at least a portion of an immunoglobulin CH2 constant region
• CH3 is at least a portion of an immunoglobulin CH3 constant region
• m, n, o, p, q, r, and s can be independently an integer between 0, 1 or 2 and 10, mimicing different types of immunoglobulin molecules, e.g., but not limited to IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, and the like, or combination thereof.
• a hinge core mimetibody ofthe present invention mimics an antibody structure with its inherent properties and functions, while providing a therapeutic peptide and its inherent or acquired in vitro, in vivo or in situ properties or activities.
• the various portions ofthe antibody and therapeutic peptide portions of at least one hinge core mimetibody ofthe present invention can vary as described herein in combinatoin with what is known in the art.
• a "hinge core mimetibody,” “hinge core mimetibody portion,” or “hinge core mimetibody fragment” and/or “hinge core mimetibody variant” and the like mimics, has or simulates at least one ligand binding or at least one biological activity of at least one protein, such as but not limited to at least one biologically active portion of at least one of SEQ ID NOS: 1-979, in vitro, in situ and/or preferably in vivo.
• a suitable hinge core mimetibody, specified portion or variant ofthe present invention can bind at least one protein ligand and includes at least one protein ligand, receptor, soluble receptor, and the like.
• a suitable hinge core mimetibody, specified portion, or variant can also modulate, increase, modify, activate, at least one protein receptor signaling or other measurable or detectable activity.
• Mimetibodies useful in the methods and compositions ofthe present invention are characterized by suitable affinity binding to protein ligands or receptors and optionally and preferably having low toxicity.
• a hinge core mimetibody where the individual components, such as the portion of variable region, constant region (without a CHI portion) and framework, or any portion thereof (e.g., a portion ofthe J, D or V rgions ofthe variable heavy or light chain;, at least one portion of at least one hinge region, the constant heavy chain or light chain, and the like) individually and/or collectively optionally and preferably possess low immunogenicity, is useful in the present invention.
• the mimetibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved.
• Low immunogenicity is defined herein as raising significant HAMA, HACA or HAHA responses in less than about 75%, or preferably less than about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, and/or 1 % ofthe patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (see, e.g., Elliott et t., Lancet 344:1125-1127 (1994)).
• the isolated nucleic acids ofthe present invention can be used for production of at least one hinge core mimetibody, fragment or specified variant thereof, which can be used to effect in an cell, tissue, organ or animal (including mammals and humans), to modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least one protein related condition, selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic disorder or disease, an anemia; an immune/autoimmune; and or an cancerous/infecteous, as well as other known or specified protein related conditions.
• Such a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms.
• the effective amount can comprise an amount of about 0.0001 to 500 mg/kg per single or multiple administration, or to achieve a serum concentration of 0.0001-5000 ⁇ g/ml serum concentration per single or multiple adminstration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts.
• the hinge core mimetibody can optionally comprise at least one CH3 region directly linked with at least one CH2 region directly linked with at least portion of at lesat one hinge region fragment (H), such as comprising at least one core hinge region, directly linked with an optional linker sequence (L), directly linked to at least one therapeutic peptide (P), optionally further directly linked with at least a portion of at least one variable antibody sequence (V).
• H hinge region fragment
• L optional linker sequence
• P therapeutic peptide
• V variable antibody sequence
• the pair can be linked by association or covalent linkage.
• a hinge core mimetibody ofthe present invention mimics an antibody structure with its inherent properties and functions, while providing a therapeutic peptide and its inherent or acquired in vitro, in vivo or in situ properties or activities.
• the various portions ofthe antibody and therapeutic peptide portions of at least one hinge core mimetibody ofthe present invention can vary as described herein in combinatoin with what is known in the art.
• Mimetibodies ofthe present invention thus provide at least one suitable property as compared to known proteins, such as, but not limited to, at least one of increased half-life, increased activity, more specific activity, increased avidity, increased or descreyse off rate, a selected or more suitable subset of activities, less immieuxicity, increased quality or duration ' of at least one desired therapeutic effect, less side effects, and the like.
• Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. For example, papain or pepsin cleavage can generate hinge core mimetibody Fab or F(ab') 2 fragments, respectively.
• Mimetibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream ofthe natural stop site.
• a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CHI domain and/or hinge region of the heavy chain.
• the various portions of mimetibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
• a nucleic acid encoding the variable and constant regions of a human antibody chain can be expressed to produce a contiguous protein for use in mimetibodies ofthe present invention. See, e.g., Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: AlZ- ⁇ l ⁇ (1988), regarding single chain antibodies.
• human mimetibody refers to an antibody in which substantially every part of the protein (e.g., therapeutic peptide, framework, C , C H domains (e.g., C H 2, C H 3), hinge, (V L , V H )) is expected to be substantially non-immunogenic, with only minor sequence changes or variations. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans relative to non-modified human antibodies, or mimetibodies ofthe prsent invention. Thus, a human antibody and corresponding hinge core mimetibody ofthe present invention is distinct from a chimeric or humanized antibody.
• a human antibody and hinge core mimetibody can be produced by a non- human animal or cell that is capable of expressing human immunoglobulins (e.g., heavy chain and/or light chain) genes, and for a hinge core mimetibody.
• Human mimetibodies that are specific for at least one protein ligand or receptor thereof can be designed against an appropriate ligand, such as isolated and/or protein receptor or ligand, or a portion thereof (including synthetic molecules, such as synthetic peptides). Preparation of such mimetibodies are performed using known techniques to identify and characterize ligand binding regions or sequences of at least one protein or portion thereof.
• At least one hinge core mimetibody or specified portion or variant ofthe present invention is produced by at least one cell line, mixed cell line, immortalized cell or clonal population of immortalized and/or cultured cells.
• Immortalized protein producing cells can be produced using suitable methods.
• the at least one hinge core mimetibody or specified portion or variant is generated by providing nucleic acid or vectors comprising DNA derived or having a substantially similar sequence to, at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement, and which further comprises a mimetibody structure as described herein, e.g., but not limited to Formula (I), wherein known portions of :C- and N-termiinal variable regions can be used for V, hinge regions for H, CH2 for CH2 and CH3 for CH3, as known in the art.
• Formula (I) wherein known portions of :C- and N-termiinal variable regions can be used for V, hinge regions for H, CH2 for CH2 and CH3 for CH3, as known in the art.
• the term "functionally rearranged,” as used herein refers to a segment of nucleic acid from an immunoglobulin locus that has undergone V(D)J recombination, thereby producing an immunoglobulin gene that encodes an immunoglobulin chain (e.g., heavy chain, light chain), or any portion thereof.
• a functionally rearranged immunoglobulin gene can be directly or indirectly identified using suitable methods, such as, for example, nucleotide sequencing, hybridization (e.g., Southern blotting, Northern blotting) using probes that can anneal to coding joints between gene segments or enzymatic amplification of immunoglobulin genes (e.g., polymerase chain reaction) with primers that can anneal to coding joints between gene segments.
• Mimetibodies, specified portions and variants ofthe present invention can also be prepared using at least one hinge core mimetibody or specified portion or variant encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such mimetibodies or specified portions or variants in their milk. Such animals can be provided using known methods as applied for antibody encoding sequences. See, e.g., but not limited to, US patent nos.
• Mimetibodies, specified portions and variants ofthe present invention can additionally be prepared using at least one hinge core mimetibody or specified portion or variant encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such mimetibodies, specified portions or variants in the plant parts or in cells cultured therefrom.
• plant cells e.g., but not limited to tobacco and maize
• transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-1 18 (1999) and references cited therein.
• transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein.
• Antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain mimetibodies (scFv's), including tobacco seeds and potato tubers.
• scFv's single chain mimetibodies
• mimetibodies, specified portions and variants ofthe present invention can also be produced using transgenic plants, according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995);
• the mimetibodies ofthe invention can bind human protein ligands with a wide range of affinities (K D ).
• at least one human hinge core mimetibody ofthe present invention can optionally bind at least one protein ligand with high affinity.
• At least one hinge core mimetibody ofthe present invention can bind at least one protein ligand with a K D equal to or less than about IO “9 M or, more preferably, with a K D equal to or less than about 0.1-9.9 (or any range or value therein) X 10 "10 M, 10 "u , IO "12 , 10 "13 or any range or value therein.
• the affinity or avidity of a hinge core mimetibody for at least one protein ligand can be determined experimentally using any suitable method, e.g., as used for determing antibody- antigen binding affinity or avidity. (See, for example, Berzofsky, et al, "Antibody- Antigen Interactions," In Fundamental Immunology, Paul, W.
• the measured affinity of a particular hinge core mimetibody- ligand interaction can vary if measured under different conditions (e.g., salt concentration, pH).
• affinity and other ligand-binding parameters e.g., K D , K a , K d
• K D , K a , K d affinity and other ligand-binding parameters
• nucleic Acid Molecules Using the information provided herein, such as the nucleotide sequences encoding at least 90-100% ofthe contiguous amino acids of at least one of SEQ ID NOS: 1-979 as well as at least one portion of an antibody, wherein the above sequences are inserted as the P sequence of Formula (I) to provide a hinge core mimetibody ofthe present invention, further comprising specified fragments, variants or consensus sequences thereof, or a deposited vector comprising at least one of these sequences, a nucleic acid molecule ofthe present invention encoding at least one hinge core mimetibody or specified portion or variant can be obtained using methods described herein or as known in the art.
• Nucleic acid molecules ofthe present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof.
• the DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.
• Isolated nucleic acid molecules ofthe present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, nucleic acid molecules comprising the coding sequence for a hinge core mimetibody or specified portion or variant; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy ofthe genetic code, still encode at least one hinge core mimetibody as described herein and/or as known in the art.
• ORF open reading frame
• nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy ofthe genetic code, still encode at least one hinge core mimetibody as described herein and/or as known in the art.
• the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific hinge core mimetibody or specified portion or variants ofthe present invention.
• nucleic acid molecules ofthe present invention which comprise a nucleic acid encoding a hinge core mimetibody or specified portion or variant can include, but are not limited to, those encoding the amino acid sequence of a hinge core mimetibody fragment, by itself; the coding sequence for the entire hinge core mimetibody or a portion thereof; the coding sequence for a hinge core mimetibody, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, intron, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functional
• sequence encoding a hinge core mimetibody or specified portion or variant can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification ofthe fused hinge core mimetibody or specified portion or variant comprising a hinge core mimetibody fragment or portion.
• a marker sequence such as a sequence encoding a peptide that facilitates purification ofthe fused hinge core mimetibody or specified portion or variant comprising a hinge core mimetibody fragment or portion.
• polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
• Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
• Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
• Low stringency conditions allow selective hybridization of sequences having about 40-99%) sequence identity and can be employed to identify orthologous or paralogous sequences.
• polynucleotides of this invention will encode at least a portion of a hinge core mimetibody or specified portion or variant encoded by the polynucleotides described herein.
• the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a hinge core mimetibody or specified portion or variant ofthe present invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference. Construction of Nucleic Acids
• the isolated nucleic acids ofthe present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well- known in the art.
• the nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention.
• a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation ofthe polynucleotide.
• translatable sequences can be inserted to aid in the isolation ofthe translated polynucleotide ofthe present invention.
• a hexa-histidine marker sequence provides a convenient means to purify the proteins ofthe present invention.
• the nucleic acid ofthe present invention - excluding the coding sequence - is optionally a vector, adapter, or linker for cloning and/or ' expression of a polynucleotide ofthe present invention.
• Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation ofthe polynucleotide, or to improve the introduction ofthe polynucleotide into a cell.
• Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. See, e.g., Ausubel, supra; or Sambrook, supra.
• RNA, cDNA, genomic DNA, or any combination thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
• oligonucleotide probes that selectively hybridize, under suitable stringency conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
• the isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).
• the isolated nucleic acids ofthe present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double- stranded DNA by hybridization with a complementary sequence, or by polymerization with a
• DNA polymerase using the single strand as a template.
• One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.
• the present invention further provides recombinant expression cassettes comprising a nucleic acid ofthe present invention.
• a nucleic acid sequence ofthe present invention for example a cDNA or a genomic sequence encoding a hinge core mimetibody or specified portion or variant ofthe present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell.
• a recombinant expression cassette will typically comprise a polynucleotide ofthe present invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription ofthe polynucleotide in the intended host cell.
• Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression ofthe nucleic acids ofthe present invention.
• isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in intron) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of a polynucleotide ofthe present invention.
• endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution, as known in the art.
• a polynucleotide ofthe present invention can be expressed in either sense or anti-sense orientation as desired.
• control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable characteristics.
• Another method of suppression is sense suppression.
• Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes.
• Vectors And Host Cells The present invention also relates to vectors that include isolated nucleic acid molecules ofthe present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one hinge core mimetibody or specified portion or variant by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
• the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced into a cell using suitable 'known methods, such as electroporation and the like, other known methods include the use of the vector as a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
• the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the DNA insert should be operatively linked to an appropriate promoter.
• the expression constructs will further contain sites optionally for at least one of transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
• the coding portion ofthe mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end ofthe mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
• Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat.Nos.
• Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dext n mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
• At least one hinge core mimetibody or specified portion or variant ofthe present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions.
• a region of additional amino acids, particularly charged amino acids, can be added to the N- terminus of a hinge core mimetibody or specified portion or variant to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
• peptide moieties can be added to a hinge core mimetibody or specified portion or variant ofthe present invention to facilitate purification. Such regions can be removed prior to final preparation of a hinge core mimetibody or at least one fragment thereof.
• COS-1 e.g., ATCC CRL 1650
• COS-7 e.g., ATCC CRL-1651
• HEK293, BHK21 e.g., ATCC CRL-10
• CHO e.g., ATCC CRL 1610
• BSC-1 e.g., ATCC CRL-26 cell lines
• hepG2 cells P3X63Ag8.653, SP2/0-Agl4, 293 cells
• HeLa cells and the like which are readily available from, for example, American Type Culture Collection, Manassas, Va.
• Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells.
• Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851).
• the recombinant cell is a P3X63Ab8.653 or a SP2/0-Agl4 cell.
• Expression vectors for these cells can include one or more ofthe following expression control sequences, such as, but not limited to an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat.Nos.
• an HSV tk promoter an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (US Pat.No. 5,266,491), at least one human immunoglobulin promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.
• nucleic acids or proteins ofthe present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
• polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
• An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing ofthe transcript can also be included.
• An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
• a hinge core mimetibody or specified portion or variant can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
• High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in
• Mimetibodies or specified portions or variants ofthe present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells.
• a eukaryotic host including, for example, yeast, higher plant, insect and mammalian cells.
• the hinge core mimetibody or specified portion or variant ofthe present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred.
• Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37- 1742, Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.
• the isolated mimetibodies ofthe present invention comprise a hinge core mimetibody or specified portion or variant encoded by any one ofthe polynucleotides ofthe present invention as discussed more fully herein, or any isolated or prepared hmge core mimetibody or specified portion or variant thereof
• the hinge core mimetibody or ligand-bmding portion or variant binds at least one protein ligand or receptor, and, thereby provides at least one biological activity ofthe corresponding protein or a fragment thereof.
• Different therapeutically or diagnostically significant proteins are well known in the art and suitable assays or biological activities of such proteins are also well known in the art.
• an embodiment of the present invention may target one or more growth factors, or, conversely, derive the target-binding moiety from one or more growth factors.
• growth factors are hormones or cytokine proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation.
• Many growth factors are quite versatile, stimulating cellular division in numerous different cell types; while others are specific to a particular cell-type.
• Table 1 presents several factors, but is not intended to be comprehensive or complete, yet introduces some ofthe more commonly known factors and their principal activities. Table 1: Growth Factors
• Additional growth factors that may be produced in accordance with the present invention include Activin (Vale et al., 321 Nature 776 (1986); Ling et al., 321 Nature 779 (1986)), Inhibin (U.S. Patent Nos. 4,737,578; 4,740,587), and Bone Morphongenic Proteins (BMPs) (U.S. Patent No. 5,846,931; Wozney, Cellular & Molecular Biology of Bone 131-167 (1993)).
• BMPs Bone Morphongenic Proteins
• the present invention may target or use other cytokines. Secreted primarily from leukocytes, cytokines stimulate both the humoral, and cellular immune responses, as well as the activation of phagocytic cells.
• lymphokines that are secreted from lymphocytes are termed lymphokines, whereas those secreted by monocytes or macrophages are termed monokines.
• a large family of cytokines are produced by various cells ofthe body.
• Many ofthe lymphokines are also known as interleukins (ILs), because they are not only secreted by leukocytes, but are also able to affect the cellular responses of leukocytes. More specifically, interleukins are growth factors targeted to cells of hematopoietic origin. The list of identified interleukins grows continuously. See, e.g., U.S. Patent No. 6,174,995; U.S. Patent No. 6,143,289; Sallusto et al., 18 Annu. Rev.
• Additional growth factor/cytokines encompassed in the present invention include pituitary hormones such as human growth hormone (HGH), follicle stimulating hormones (FSH, FSH ⁇ , and FSH ⁇ ), Human Chorionic Gonadotrophins (HCG, HCGcc, HCG ⁇ ), uFSH (urofollitropin), Gonatropin releasing hormone (GRH), Growth Hormone (GH), leuteinizing hormones (LH, LH ⁇ , LH ⁇ ), somatostatin, prolactin, thyrotropin (TSH, TSH ⁇ , TSH ⁇ ), thyrotropin releasing hormone (TRH), parathyroid hormones, estrogens, progesterones, testosterones, or structural or functional analog thereof.
• cytokine family also includes tumor necrosis factors, colony stimulating factors, and interferons. See, e.g., Cosman, 7 Blood Cell (1996); Grass et al., 85 Blood 3378 (1995); Beutler et al., 7 Annu. Rev. Immunol. 625 (1989); Aggarwal et al., 260 J. Biol. Chem. 2345 (1985); Pennica et al., 312 Nature 724 (1984); R & D Systems, Cytokine Mini-Reviews, at http://www.rndsystems.com. Several cytokines are introduced, briefly, in Table 2 below.
• cytokines of interest include adhesion molecules (R & D Systems, Adhesion Molecule I (1996), at http://www.rndsystems.com); angiogenin (U.S. Patent No. 4,721,672; Moener et al., 226 Eur.
• MSP Macrophage Stimulating Protein
• Neurotrophic Factors U.S. Patent Nos. 6,005,081; 5,288,622;
• Pleiotrophin/Midkine (PTN/MK) (Pedraza et al., 117 J. Biochem. 845 (1995); Tamura et al , 3
• cytokines proteins or chemical moieties that interact with cytokines, such as Matrix Metalloproteinases (MMPs) (U.S. Patent No. 6,307,089; Nagase,
• Nitric Oxide Synthases (Fukuto, 34 Adv. Pharm 1 (1995); U.S. Patent No. 5,268,465).
• the present invention may also be used to affect blood proteins, a generic name for a vast group of proteins generally circulating in blood plasma, and important for regulating coagulation and clot dissolution. See, e.g , Haematologic Technologies, Inc., HTI CATALOG, at www.haemtech.com. Table 3 introduces, in a non-limiting fashion, some ofthe blood proteins contemplated by the present invention.
• Fibrinogen Plasma fibrinogen a large glycoprotein, FURLAN, Fibrinogen, INHUMAN disulfide linked dimer made of 3 pairs PROTEIN DATA, (Haeberli, ed., VCH of non-identical chains (Aa, Bb and g), Publishers, N.Y., 1995); Doolittle, in made in liver.
• Aa has N-terminal peptide HAEMOSTASIS & THROMBOSIS, 491-513 (fibrinopeptide A (FPA), factor XHIa (3rd ed., Bloom et al., eds., Churchill crosslinking sites, and 7 Livingstone, 1994); HANTGAN, et al., in phosphorylation sites.
• Bb has HAEMOSTASIS & THROMBOSIS 269-89 fibrinopeptide B (FPB), 1 of 3 N-linked (2d ed., Forbes et al., eds., Churchill carbohydrate moieties, and an N- Livingstone, 1991). terminal pyroglutamic acid.
• the g chain contains the other N-linked glycos. site, and factor Xllla cross-linking sites.
• Two elongated subunits ((AaBbg) 2 ) align in an antiparallel way forming a trinodular arrangement ofthe 6 chains. Nodes formed by disulfide rings between the 3 parallel chains.
• Central node (n- disulfide knot, E domain) formed by N- termini of all 6 chains held together by 11 disulfide bonds, contains the 2 Ila- sensitive sites. Release of FPA by cleavage generates Fbn I, exposing a polymerization site on Aa chain. These sites bind to regions on the D domain of Fbn to form proto-fibrils.
• Additional blood proteins contemplated herein include the following human seram proteins, which may also be placed in another category of protein (such as hormone or antigen): Actin, Actinin, Amyloid Serum P, Apolipoprotein E, B2-Microglobulin, C-Reactive Protein (CRP), Cholesterylester transfer protein (CETP), Complement C3B, Ceraplasmin, Creatine Kinase, Cystatin, Cytokeratin 8, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, Cytokeratin 20, Desmin, Desmocollin 3, FAS (CD95), Fatty Acid Binding Protein, Ferritin, Filamin, Glial Filament Acidic Protein, Glycogen Phosphorylase Isoenzyme BB (GPBB), Haptoglobulin, Human Myoglobin, Myelin Basic Protein, Neurof ⁇ lament, Placental Lactogen, Human SHBG, Human Thyroid Peroxidase, Receptor Associated Protein
• Prealbumin Albumin, Alpha-1-Acid Glycoprotein, Alpha- 1 -Anti chymotrypsin, Alpha-1- Antitrypsin, Alpha-Fetoprotein, Alpha- 1-Microglobulin, Beta-2-microglobulin, C-Reactive Protein, Haptoglobulin, Myoglobulin, Prealbumin, PSA, Prostatic Acid Phosphatase, Retinol Binding Protein, Thyroglobulin, Thyroid Microsomal Antigen, Thyroxine Binding Globulin, Transferrin , Troponin I, Troponin T, Prostatic Acid Phosphatase, Retinol Binding Globulin
• the target in the present invention may also incorporate or target neurotransmitters, or functional portions thereof.
• Neurotransmitters are chemicals made by neurons and used by them to transmit signals to the other neurons or non-neuronal cells (e.g., skeletal muscle; myocardium, pineal glandular cells) that they innervate. Neurotransmitters produce their effects by being released into synapses when their neuron of origin fires (i.e., becomes depolarized) and then attaching to receptors in the membrane of the post-synaptic cells.
• Neurotransmitters can also produce their effects by modulating the production of other signal-transducing molecules ("second messengers") in the post-synaptic cells. See generally COOPER, BLOOM & ROTH, THE BIOCHEMICAL BASIS OF NEUROPHARMACOLOGY (7th Ed. Oxford Univ. Press, NYC, 1996); http://web.indstate.edu/thcme/mwking/nerves. Neurotransmitters contemplated in the present invention include, but are not limited to,
• peptides may be used in conjunction with the present invention.
• peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL- Ira, leptin, CTLA4, TRAIL, TGF- , and TGF- ⁇ .
• Peptide antagonists are also of interest, particularly those antagonistic to the activity of TNF, leptin, any ofthe interleukins (IL-1 - IL-23, etc.), and proteins involved in complement activation (e.g., C3b).
• Targeting peptides are also of interest, including tumor-homing peptides, membrane-transporting peptides, and the like.
• peptides may be prepared by methods disclosed and/or known in the art. Single letter amino acid abbreviations are used in most cases.
• the X in these sequences (and throughout this specification, unless specified otherwise in a particular instance) means that any ofthe 20 naturally occurring amino acid residues or know derivatives thereof may be present, or any know modified amino acid thereof. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers, and a few tandemlinked examples are provided in the table. Linkers are listed as " ⁇ " and may be any of the linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity.
• Any peptide containing a cysteinyl residue may optionally be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle.
• a few crosslinked examples are provided in the table.
• Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well; see, for example, EPO-mimetic peptides in Table 5.
• a few examples of intrapeptide disulfide-bonded peptides are specified in the table. Any of these peptides may be derivatized as described herein, and a few derivatized examples are provided in the table.
• the capping amino group is shown as -NFL.
• the substitutions are denoted by a ⁇ , which signifies any ofthe moieties known in the art, e.g., as described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), J. Med. Chem. 40:2876-82, which are entirely incorporated by reference.
• the J substituent and the Z substituents (Z 5 , Z 6 , ... Z 40 ) are as defined in U.S. Pat. Nos.
• Xaa and Yaa below are as defined in WO 98/09985, published March 12,1998, which is entirely incorporated herein by reference.
• AAj, AA 2 , AB), AB 2 , and AC are as defined in International application WO 98/53842, published December 3, 1998, which is entirely incorporated by reference.
• X 1 , X 2 , X 3 , and X 4 in Table 18 only are as, defined in European application EP 0 911 393, published April 28,1999, entirely incorporated herein by reference.
• Residues appearing in boldface are D- amino acids, but can be optionally L-amino acids. All peptides are linked through peptide bonds unless otherwise noted. Abbreviations are listed at the end of this specification. In the "SEQ ID NO.” column, "NR" means that no sequence listing is required for the given sequence.
• FEWTPGWYQJY 58 AcFEWTPGWYQJY I 59 FEVffPGWpYQJY 60 FAWTPGYWQJY 61 FEWAPGYWQJY 62
• FEWVPGYWQJY 63 FEWTPGYWQJY 64 AcFEWTPGYWQJY 65 FEWTPaWYQJY 66 FEWTPSarWYQJY 67 FEWTPGYYQPY 68 FEWTPGWWQPY 69
• FAWTPGYWQJY 80 FEWAPGYWQJY 81 FEWVPGYWQJY 82 FEWTPGYWQJY 83 AcFEWTPGYWQJY 84 FEWTPAWYQJY 85 FEWTPSarWYQJY 86
• WIEWWQPYSVQS 121 SLIY QPYSLQM 122 TRLYWQPYSVQR 123 RCDYWQPYSVQT 124 MRVFWQPYSVQN 125
• VGRWYQPYSVQR 143 VHVYWQPYSVQR 144 QARWYQPYSVQR 145 VHVYWQPYSVQT 146 RSVYWQPYSVQR 147
• RLVYWQPYSVQA 160 SRVWYQPYAKGL 161 SRVWYQPYAQGL 162 SRVWYQPYAMPL 163 SRVWYQPYSVQA 164
• DPLFWQPYALPL 172 SRQWVQPYALPL 173 IRSWWQPYALPL 174 RGYWQPYALPL 175 RLLWVQPYALPL 176
• IWYQPYAMPL 199 SNMQPYQRLS 200 TFVYWQPYAVGLPAAETACN 201 TFVYWQPYSVQMTITGKVTM 202 TFVYWQPYSSHXXVPXGFPL 203 TFVYWQPYYGNPQWAIHVRH 204 TFVYWQPYVLLELPEGAVRA 205
• WEQNVYWQPYSVQSFAD 224 SDVVYWQPYSVQSLEM 225 YYDGVYWQPYSVQVMPA 226 SDIWYQPYALPL 227 QRIWWQPYALPL 228
• E ⁇ MFWQPYALPL 235 DYVWQQPYALPL 236 MDLLVQWYQPYALPL 237 ' GSKVILWYQPYALPL 238 RQGANIWYQPYALPL 239 GGGDEPWYQPYALPL 240
• SQLERTWYQPYALPL 241 ETWVREWYQPYALPL 242 KKGSTQWYQPYALPL 243 LQARMNWYQPYALPL 244 EPRSQKWYQPYALPL 245
• VKQKWRWYQPYALPL 246 LRRHDVWYQPYALPL 247 RSTASIWYQPYALPL 248 ESKEDQWYQPYALPL 249 EGLTMKWYQPYALPL 250
• EGSREGWYQPYALPL 251 VIEWWQPYALPL 252 VWYWEQPYALPL 253 ASEWWQPYALPL 254 FYEWWQPYALPL 255 EGWWVQPYALPL 256 WGEWLQPYALPL 257
• DYVWEQPYALPL 258 AHTWWQPYALPL 259 FIEWFQPYALPL 260 WLAWEQPYALPL 261 VMEWWQPYALPL 262
• AFYQPYALPL 292 FLYQPYALPL 293 VCKQPYLEWC 294 ETPFTWEESNAYYWQPYALPL 295 QGWLTWQDSVDMYWQPYALPL 296
• SDAFTTQDSQAMYWQPYALPL 309 GDDAAWRTDSLTYWQPYALPL 310
• AIIRQLYRWSEMYWQPYALPL 311 ENTYSPNWADSMYWQPYALPL ' 312 MNDQTSEVSTFPYWQPYALPL 313
• ADVLYWQPYAPVTLWV 343 GDVAEYWQPYALPLTSL 344 SWTDYGYWQPYALPISGL 345 FEWTPGYWQPYALPL 346 FEWTPGYWQJYALPL 347
• VYWQPYSVQ 394 VY-Nap-QPYSVQ 395 TFVYWQJYALPL 396 FEWTPGYYQJ-Bpa 397 XaaFEWTPGYYQJ-Bpa 398
• RLVWFQPYSVQR 411 RLVYWQPYSIQR 412 DNSSWYDSFLL 413 DNTAWYESFLA 414 DNTAWYENFLL 415
• YIPFTWEESNAYYWQPYALPL 433 DGYDRWRQSGERYWQPYALPL 434 pY-INap-pY-QJYALPL 435 TANVSSFEWTPGYWQPYALPL 436 FEWTPGYWQJYALPL 437
• TANVSSFEWTPGYWQPYALPL 450 AcFEWTPGYWQJY 451 AcFEWTPGWYQJY 452 AcFEWTPGYYQJY 453 AcFEWTPAYWQJY 454
• FCVSNDRCY 534 YCRKELGQVCY 535 YCKEPGQCY 536 YCRKEMGCY 537 FCRKEMGCY 538
• CWDDGWMC 561 CSWDDGWLC 562 CPDDLWWLC 563 NGR NR GSL NR RGD NR CGRECPRLCQSSC 564
• RTDLDSLRTYTL 572 RTDLDSLRTY 573 RTDLDSLRT 574 RTDLDSLR 575 GDLDLLKLRLTL 576 GDLHSLRQLLSR 577 RDDLHMLRLQLW 578
• GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ 610 GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE 611
• GFFALIPKIISSPLFKTLLSAV 613 KGFFALIPKIISSPLFKTLLSAV 614 KKGFFALIPKIISSPLFKTLLSAV 615 KKGFFALIPKIISSPLFKTLLSAV 616 GFFALIPKIIS 617
• GIGAVLKVLTTGLPALISWIKRKRQQ 618 GIGAVLKVLTTGLPALISWIKRKRQQ 619
• GIGAVLKVLTTGLPALISWIKRKRQQ 620 GIGAVLKVLTTGLPALISWIKR 621 AVLKVLTTGLPALISWIKR 622 KLLLLLKLLK 623 KLLLKLLLKLLK 624
• KLLLKLLK 630 KLLLKLKLLK 631 KLLLKLKLLK 632 KLLLKLKLLK 633 KAAAKAAAKAAK 634
• KVVVKVVVKVKVK 635 KVVVKVKVKVK 636 KVVVKVKVKVK 637 KVVVKVKVKVK 638 KLILKL 639 KVLHLL 640 LKLRLL 641
• KPLHLL 642 KLILKLVR 643 KVFHLLHL 644 HKFRILKL 645 KPFHILHL 646
• HIGIKAHVRIIRVHII 669 RIYVKIHLRYIKKIRL 670 KIGHKARVHIIRYKII 671 RIYVKPHPRYIKKIRL 672 KPGHKARPHIIRYKII 673
• KIGWKLRVRIIRVKIGRLR 676 KINIWRmLIWWRKIVKVKRIR 677 RFAVKIRLRIIKKIRLIKKIRKRVIK 678 KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 679 RIYVKPHPRYIKKIRL 680
• KPIHKARPTIIRYKMI 686 cyclicCKGFFALIPKIISSPLFKTLLSAVC 687 CKKGFFALIPKIISSPLFKTLLSAVC 688 CKKKGFFALIPKIISSPLFKTLLSAVC 689 CyclicCRWIRIWRLIRIRC 690 CyclicCKPGHKARPHIIRYKIIC 691 CyclicCRFAVKIRLRIIKKIRLIKKIRKRVIKC 692
• KKYLNSIL 749 KKKYLD 750 cyclicCKKYLC 751 CKKYLK 752 KKYA 753 WWTDTGLW 754 WWTDDGLW 755
• KLWSEQG ⁇ WMGE 762 CWSMHGLWLC 763 GCWDNTGIWVPC 764 DWDTRGLWVY 765
• ⁇ NLKALAALAKKIL 806 KIWSILAPLGTTLVKLVA 807 LKKLLKLLKKLLKL 808 LKWKKLLKLLKKLLKKLL 809
• AESLPTLTSILWGKESV 906 AETLFMDLWHDKHILLT 907 AEILNFPLWHEPLWSTE 908 AESQTGTLNTLFWNTLR 909 AEPVYQYELDSYLRSYY 910
• STGGFDDVYDWARGVSSALTTTLVATR 940 Vinculin-binding STGGFDDVYDWARRVSSALTTTLVATR 941 Vinculin-binding SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 942 Vinculin-binding SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 943 Vinculin-binding SSPSLYTQFLVNYESAATRIQDLLIASRPSR 944 Vinculin-binding
• the present invention is also particularly useful with peptides having activity in treatment of: a VEGF related condition, e.g., but not limited to, cancer, wherein the peptide is a VEGF-mimetic or a VEGF receptor antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the like; asthma, wherein the protein of interest is a CKR3 antagonist, an IL-5 receptor antagonist, and the like; thrombosis, wherein the protein of interest is a GPIIb antagonist, a GPIIIa antagonist, and the like; autoimmune diseases and other conditions involving immune modulation, wherein the protein of interest is an IL-2 receptor antagonist, a CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin mimetic and the like.
• a VEGF related condition e.g., but not limited to, cancer
• the peptide is a VEGF-mimetic or a VEGF receptor antagonist
• EPO biological activities are well known in the art. See, e.g., Anagnostou A et al Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proceedings of the National Academy of Science (USA) 87: 5978-82 (1990); Fandrey J and Jelkman WE Interleukin 1 and tumor necrosis factor-alpha inhibit erythropoietin production in vitro. Annals ofthe New York Academy of Science 628: 250-5 (1991); Geissler K et al Recombinant human erythropoietin: A multipotential hemopoietic growth factor in vivo and in vitro. Contrib. Nephrol.
• EPO can be assayed by employing cell lines such as HCD57 , NFS-60 , TF-1 and UT-7 , which respond to the factor . EPO activity can be assessed also in a Colony formation assay by determining the number of CFU-E from bone marrow cells.
• An alternative and entirely different detection method is RT-PCR quantitation of cytokines.
• a hinge core mimetibody, or specified portion or variant thereof, that partially or preferably substantially provides at least one biological activity of at least one protein or fragment, can bind the protein or fragment ligand and thereby provide at least one activity that is otherwise mediated through the binding of protein to at least one protein ligand or receptor or through other protein-dependent or mediated mechanisms.
• hinge core mimetibody activity refers to a hinge core mimetibody that can modulate or cause at least one protein-dependent activity by about 20-10,000%, preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 % or more depending on the assay.
• a human hinge core mimetibody or specified portion or variant ofthe invention can be similar to any class (IgG, IgA, IgM, etc.) or isotype and can comprise at least a portion of a kappa or lambda light chain.
• the human hinge core mimetibody or specified portion or variant comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
• the human protein human hinge core mimetibody or specified portion or variant thereof comprises an IgGl heavy chain and an IgGl light chain.
• At least one hinge core mimetibody or specified portion or variant ofthe invention binds at least one specified ligand specific to at least one protein, subunit, fragment, portion or any combination thereof.
• the at least one therapeutic peptide portion (P) of at least one mimetibody of the invention can optionally bind at least one specified ligand epitope ofthe ligand.
• the binding epitope can comprise any combination of at least one amino acid sequence of at least 1-3 amino acids to the entire specified portion of contiguous amino acids ofthe sequences selected from the group consisting of a protein ligand, such as a receptor or portion thereof.
• the hinge core mimetibody can comprise at least one N terminal heavy or light chain variable region having a defined amino acid sequence.
• Mimetibodies that bind to human protein ligands or receptors and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al, IntJMol.
• the hinge core mimetibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
• the invention also relates to mimetibodies, ligand-binding fragments, immunoglobulin chains comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
• mimetibodies or ligand-binding fragments and mimetibodies comprising such chains can bind human protein ligands with high affinity (e.g., K D less than or equal to about IO "9 M).
• Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
• a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those ofthe first amino acid.
• Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K).
• Amino Acid Codes The amino acids that make up mimetibodies or specified portions or variants ofthe present invention are often abbreviated.
• amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994), as presented in the following Table 22: TABLE 22
• a hinge core mimetibody or specified portion or variant ofthe present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
• Such or other sequences that can be used in the present invention include, but are not limited to but are not limited to the following sequences presented in Table 23, as further described in Figures 1-42 of US provisional application 60/507,349, filed 30/03/2003, entirely inco ⁇ orated by reference herein, corresponding to Figures 1-41 of PCT Appl. No. US04/19783, filed June 17, 2004, entirely inco ⁇ orated herein by reference, with corresponding SEQ ID NOS:31-72.
• Figures 1-42 SEQ ID OS:31-72
• Figures 1-41 of PCT US04/19783 show examples of heavy/light chain variable/constant region sequences, frameworks/subdomains and substitutions, portions of which can be used in Ig derived proteins ofthe present invention, as taught herein.
• the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above.
• the number of amino acid substitutions, insertions or deletions for at least one of a hinge core mimetibody or fragment e.g., but not limited to, at least one variable, constant, light or heavy chain, or Ig will not be more than 40, 30, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids, such as 1-30 or any range or value therein, as specified herein.
• a hinge core mimetibody ofthe present invention is based on the use ofthe formula I ofthe present invention, ((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portion of an N-terminus of an immunoglobulin variable region, P is at least one bioactive peptide, L is at least one linker polypeptide H is at least one portion of at least one immunoglobulin hinge region, CH2 is at least a portion of an immunoglobulin CH2 constant region, CH3 is at least a portion of an immunoglobulin CH3 constant region, m, n, o, p, q, r and s are independently an integer between 0, 1 or 2 and 10, mimicing different types of immunoglobulin molecules, e.g., but not limited to IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, and
• the optional N-terminal V portion can comprise 1-20 amino acids of at least one heavy chain variable framework 1 (FR1) region, e.g., as presented in Figures 1-9 (SEQ ID NOS:31-39) or at least one LC variable region, e.g., as presented in Figures 10-31 (SEQ ID NOS:40-61), each of such figures of US provisional application 60/507,349, filed 30/03/2003, entirely inco ⁇ orated by reference herein, corresponding to Figures 1-41 of PCT Appl. No.
• FR1 heavy chain variable framework 1
• variable sequences that comprise the sequence Q-X-Q.
• the P portion can comprise at least one any therapeutic peptide as known in the art or as described herein, such as, but not limited to those presented in Tables 1-21, SEQ ID NOS:l- 979, or as known in th e art, or any combination or consensus sequence thereof, or any fusion protein thereof.
• the optional linker sequence can be any suitable peptide linker as known in the art.
• Preferred sequence include any combination of G and S, e.g., Xl-X2-X3-X4-Xn, where X can be G or S, and n can be 5-30.
• Non-limiting examples include, GS, GGGS, GSGGGS, GSGGGSGG, and the like.
• the CHI portion is not used and a variable number of amino acids from the N-terminus ofthe hinge region are deleted, e.g., as referenced to Figures 1-42 of US provisional application 60/507,349, filed 30/03/2003, entirely inco ⁇ orated by reference herein, corresponding to Figures 1-41 of PCT Appl. No. US04/19783, filed June 17, 2004, entirely inco ⁇ orated herein by reference, and Table 3.
• variable number of amino acids used for the hinge core portion of a mimetibody ofthe present invention include, but are not limited to, deletion of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 1-3, 2-5, 2-7, 2-8, 3-9, 4-10, 5-9, 5-10, 5-15, 10- 20, 2-30, 20-40, 10-50, or any range or value therein, ofthe N-terminal amino acids of at least one hinge region, e.g., as presented in Figures 32-40 of US provisional application 60/507,349, filed 30/03/2003, entirely inco ⁇ orated by reference herein, corresponding to Figures 1-41 of
• a hinge core regions ofthe present invention includes a deletion ofthe N-terminous ofthe hinge region to provide a hinge core region that includes a deletion up to but not including a Cys residue or up to but not including a sequence Cys-Pro- Xaa-Cys.
• such hinge core sequences used in a hinge coi e mimetibody ofthe present invention include amino acids 109-113 or 112-113 of Fig. 36 (SEQ ID NO:66) (IgGl); 105-110 or 109-110 of Fig. 37 (SEQ ID NO:67) (IgG2); 111-160, 114-160, 120-160, 126-160, 129-160, 135-160, 141-160, 144-160, 150-160, 156-160 and 159-160 of Fig. 38 (SEQ ID NO:68) (IgG3); or 106-110 or 109-110 of Fig.
• CH2, CH3 and optional CH4 sequence can be any suitable human or human compatable sequence, e.g., as presented in Figures 1-41 and Table 23 of US provisional application 60/507,349, filed 30/03/2003, entirely incorporated by reference herein, corresponding to Figures 1-41 of PCT Appl. No.
• the resulting mutant molecules are then tested for biological activity, such as, but not limited to at least one protein related activity, as specified herein or as known in the art.
• Sites that are critical for hinge core mimetibody or specified portion or variant binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).
• Mimetibodies or specified portions or variants ofthe present invention can comprise as P portion of Formula (I), but are not limited to, at least one portion, sequence or combination selected from 3 to all the of at least one of SEQ ID NOS: 1-979.
• Non-limiting variants that can enhance or maintain at least one ofthe listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution, insertion or deletion that does not significantly affect the suitable biological activtities or functions of said hinge core mimetibody.
• a hinge core mimetibody or specified portion or variant can further optionally comprise at least one functional portion of at least one polypeptide as P portion of Formula (I), at least one of 90-100% of SEQ ID NOS: 1-979.
• A. hinge core mim tibody can further optionally comprise an amino acid sequence for the P portion of Formula (I), selected from one or more of SEQ ID NOS: 1-979.
• the P amino acid sequence of an immunoglobulin chain, or portion thereof has about 90-100% identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the corresponding amino acid sequence of the corresponding portion of at least one of SEQ ID NOS: 1-979.
• 90-100% amino acid identity i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein
• 90-100% amino acid identity is determined using a suitable computer algorithm, as known in the art.
• Mimetibodies or specified portions or variants ofthe present invention can comprise any number of contiguous amino acid residues from a hinge core mimetibody or specified portion or variant ofthe present invention, wherein that number is selected from the group of integers consisting of from 10-100% ofthe number of contiguous residues in a hinge core mimetibody.
• this subsequence of contiguous amino acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein.
• the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more.
• the present invention includes at least one biologically active hinge core mimetibody or specified portion or variant of the present invention.
• Biologically active mimetibodies or specified portions or variants have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%- 1000% of that ofthe native (non-synthetic), endogenous or related and known inserted or fused protein or specified portion or variant. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.
• the invention relates to human mimetibodies and ligand-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety.
• the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
• the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
• the modified mimetibodies and ligand-binding fragments ofthe invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the hinge core mimetibody or specified portion or variant.
• Each organic moiety that is bonded to a hinge core mimetibody or ligand-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
• fatty acid encompasses mono-carboxylic acids and di-carboxylic acids.
• Hydrophilic polymers suitable for modifying mimetibodies of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
• polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
• carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
• polymers of hydrophilic amino acids e.g., poly
• the hydrophilic polymer that modifies the hinge core mimetibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
• PEG2500, PEG 50 oo, PEG 75 oo, PEG 90 oo, PEGioooo, PEGi 25 oo, PEG ⁇ 50 oo, and PEG 2 o,ooo, wherein the subscript is the average molecular weight ofthe polymer in Daltons can be used.
• the hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups.
• Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods.
• a polymer comprising an amine group can be coupled to a carboxylate ofthe fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
• Fatty acids and fatty acid esters suitable for modifying mimetibodies ofthe invention can be saturated or can contain one or more units of unsaturation.
• Fatty acids that are suitable for modifying mimetibodies ofthe invention include, for example, n-dodecanoate ( 2 , laurate), n-tetradecanoate (C ] , myristate), n-octadecanoate (C ]8 , stearate), n-eicosanoate (C 2 o, arachidate), n-docosanoate (C 22 , behenate), n-triacontanoate (C 30 ), n-tetracontanoate (C 0 ), cis- ⁇ 9-octadecanoate (C ]8 , oleate), all cis- ⁇ 5,8,11,14-eicosatetraenoate (C 2 o, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedi
• Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group.
• the lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms.
• the modified human mimetibodies and ligand-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents.
• activating group is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
• amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.
• Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB- thiol), and the like.
• An aldehyde functional group can be coupled to amine- or hydrazide- containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
• Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
• An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent -C 12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
• Suitable linker moieties include, for example, tetraethylene glycol, -(CH 2 ) 3 -, -NH-(CH 2 ) 6 -NH-, -(CH 2 ) 2 -NH- and -CH 2 -0-CH 2 - CH 2 -0-CH 2 -CH 2 -0-CH-NH-.
• Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc- ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.
• a mono-Boc-alkyldiamine e.g., mono-Boc- ethylenediamine, mono-Boc-diaminohexane
• EDC l-ethyl-3-(3- dimethylaminopropyl) carbodiimide
• the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative ofthe fatty acid.
• TFA trifluoroacetic acid
• the modified mimetibodies ofthe invention can be produced by reacting an human hinge core mimetibody or ligand-binding fragment with a modifying agent.
• the organic moieties can be bonded to the hinge core mimetibody in a non-sit specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
• Modified human mimetibodies or ligand-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of a hinge core mimetibody or ligand-binding fragment. The reduced hinge core mimetibody or ligand-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified hinge core mimetibody ofthe invention.
• Modified human mimetibodies and ligand-binding fragments comprising an organic moiety that is bonded to specific sites of a hinge core mimetibody or specified portion or variant of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et al, Protein Sci. 6(10):2233-2241 (1997); Itoh et al, Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al, Biotechnol.
• suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et al, Protein Sci. 6(10):2233-2241 (1997); Itoh et
• the present invention also provides at least one hinge core mimetibody or specified portion or variant composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more mimetibodies or specified portions or variants thereof, as described herein and/or as known in the art that are provided in a non-naturally occurring composition, mixture or form.
• Such composition percentages are by weight, volume, concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein.
• compositions can comprise 0.00001-99.9999 percent by weight, volume, concentration, molarity, or molality as liquid, gas, or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein, on any range or value therein, such as but not limited to 0.00001, 0.00003, 0.00005, 0.00009, 0.0001, 0.0003, 0.0005, 0.0009, 0.001,
• compositions ofthe present invention thus include but are not limited to 0.00001-100 mg/ml and/or 0.00001-100 mg/g.
• the composition can optionally further comprise an effective amount of at least one compound or protein selected from at least one of an anti -infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplactic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a nutritional drug or the like.
• CV cardiovascular
• CNS central nervous system
• ANS autonomic nervous system
• GI gastrointestinal
• Such drugs are well known in the art, including formulations, indications, dosing and administration for each presented herein (see, e.g., Nursing 2001 Handbook of Drugs, 21 st edition, Springhouse Co ⁇ ., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT, each entirely inco ⁇ orated herein by reference).
• the anti-infective drug can be at least one selected from amebicides or at least one antiprotozoals, anthelmintics, antifungals, antimalarials, antituberculotics or at least one antileprotics, aminoglycosides, penicillins, cephalosporins, tetracyclines, sulfonamides, fluoroquinolones, antivirals, macrolide anti-infectives, miscellaneous anti-infectives.
• the CV drug can be at least one selected from inotropics, antiarrhythmics, antianginals, antihypertensives, antilipemics, and miscellaneous cardiovascular drugs.
• the CNS drug can be at least one selected from nonnarcotic analgesics or at least one selected from antipyretics, nonsteroidal anti-inflammatory drugs, narcotic or at least one opiod analgesics, sedative- hypnotics, anticonvulsants, antidepressants, antianxiety drugs, antipsychotics, central nervous system stimulants, antiparkinsonians, miscellaneous central nervous system drugs.
• the ANS drug can be at least one selected from cholinergics (parasympathomimetics), anticholinergics, adrenergics (sympathomimetics), adrenergic blockers (sympatholytics), skeletal muscle relaxants, neuromuscular blockers.
• the respiratory tract drug can be at least one selected from antihistamines, bronchodilators, expectorants or at least one antitussives, miscellaneous respiratory drugs.
• the GI tract drug can be at least one selected from antacids or at least one adsorbents or at least one antiflatulents, digestive enzymes or at least one gallstone solubilizers, antidiarrheals, laxatives, antiemetics, antiulcer drugs.
• the hormonal drug can be at least one selected from corticosteroids, androgens or at least one anabolic steroids, estrogens or at least one progestins, gonadotropins, antidiabetic drugs or at least one glucagon, thyroid hormones, thyroid hormone antagonists, pituitary hormones, parathyroid-like drugs.
• the drug for fluid and electrolyte balance can be at least one selected from diuretics, electrolytes or at least one replacement solutions, acidifiers or at least one alkalinizers.
• the hematologic drug can be at least one selected from hematinics, anticoagulants, blood derivatives, thrombolytic enzymes.
• the antineoplastics can be at least one selected from alkylating drugs, antimetabolites, antibiotic antineoplastics, antineoplastics that alter hormone balance, miscellaneous antineoplastics.
• the immunomodulation drug can be at least one selected from immunosuppressants, vaccines or at least one toxoids, antitoxins or at least one antivenins, immune serums, biological response modifiers.
• the ophthalmic, otic, and nasal drugs can be at least one selected from ophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics, mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics, otics, nasal drugs.
• the topical drug can be at least one selected from local anti-infectives, scabicides or at least one pediculicides, topical corticosteroids.
• the nutritional drug can be at least one selected from vitamins, minerals, or calorics. See, e.g., contents of Nursing 2001 Drug Handbook, supra.
• the at least one amebicide or antiprotozoal can be at least one selected from atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, pentamidine isethionate.
• the at least one anthelmintic can be at least one selected from mebendazole, pyrantel pamoate, thiabendazole.
• the at least one antifungal can be at least one selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, terbinafine hydrochloride.
• the at least one antimalarial can be at least one selected from chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, ⁇ mefloquine hydrochloride, primaquine phosphate, pyrimethamine, pyrimethamine with sulfadoxine.
• the at least one antituberculotic or antileprotic can be at least one selected from clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, streptomycin sulfate.
• the at least one aminoglycoside can be at least one selected from amikacin sulfate, gentamicin sulfate, neomycin sulfate, streptomycin sulfate, tobramycin sulfate.
• the at least one penicillin can be at least one selected from amoxcillin/clavulanate potassium, amoxicillin trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin sodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium, mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillin disodium, ticarcillin disodium clavulanate potassium.
• the at least one cephalosporin can be at least one selected from at least one of cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium, cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime, cef ⁇ tibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate, cephradine, loracarbef.
• the at least one tetracycline can be at least one selected from demeclocycline hydrochloride, doxycycline calcium, doxycycline hyclate, doxycycline hydrochloride, doxycycline monohydrate, minocycline hydrochloride, tetracycline hydrochloride.
• the at least one sulfonamide can be at least one selected from co-trimoxazole, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfisoxazole acetyl.
• the at least one fluoroquinolone can be at least one selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate.
• the at least one fluoroquinolone can be at least one selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate.
• the at least one antiviral can be at least one selected from abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, zidovudine.
• the at least one macroline anti-infective can be at least one selected from azithromycin, clarithromycin, dirithromycin, erythromycin base, erythromycin estolate, erythromycin ethylsuccinate, erythromycin lactobionate, erythromycin stearate.
• the at least one miscellaneous anti-infective can be at least one selected from aztreonam, bacitracin, chloramphenicol sodium sucinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, imipenem and cilastatin sodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystals, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, vancomycin hydrochloride. (See, e.g., pp.
• the at least one inotropic can be at least one selected from amrinone lactate, digoxin, milrinone lactate.
• the at least one antiarrhythmic can be at least one selected from adenosine, amiodarone hydrochloride, atropine sulfate, bretylium tosylate, diltiazem hydrochloride, disopyramide, disopyramide phosphate, esmolol hydrochloride, flecainide acetate, ibutilide fumarate, lidocaine hydrochloride, mexiletine hydrochloride, moricizine hydrochloride, phenytoin, phenytoin sodium, procainamide hydrochloride, propafenone hydrochloride, propranolol hydrochloride, quinidine bisulfate, quinidine gluconate, quinidine polygalacturonate, quinidine sulfate, sotalol
• the at least one antianginal can be at least one selected from amlodipidine besylate, amyl nitrite, bepridil hydrochloride, diltiazem hydrochloride, isosorbide dinitrate 5 isosorbide mononitrate, nadolol, nicardipine hydrochloride, nifedipine, nitroglycerin, propranolol hydrochloride, verapamil, verapamil hydrochloride.
• the at least one antihypertensive can be at least one selected from acebutolol hydrochloride, amlodipine besylate, atenolol, benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate, candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol, clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride, doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate, felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate, guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride, irbe
• the at least one miscellaneous CV drug can be at least one selected from abciximab, alprostadil, arbutamine hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole, eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine hydrochloride, tirofiban hydrochloride. (See, e.g., pp. 215-336 of Nursing 2001 Drug
• the at least one nonnarcotic analgesic or antipyretic can be at least one selected from acetaminophen, aspirin, choline magnesium trisalicylate, diflunisal, magnesium salicylate.
• the at least one nonsteroidal anti-inflammatory drug can be at least one selected from celecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, sulindac.
• the at least one narcotic or opiod analgesic can be at least one selected from alfentanil hydrochloride, bupreno ⁇ hine hydrochloride, buto ⁇ hanol tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl transdermal system, fentanyl transmucosal, hydromo ⁇ hone hydrochloride, meperidine hydrochloride, methadone hydrochloride, mo ⁇ hine hydrochloride, mo ⁇ hine sulfate, mo ⁇ hine tartrate, nalbuphine hydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymo ⁇ hone hydrochloride, pentazocine hydrochloride, pentazocine hydrochloride and naloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride, propoxyphene napsylate, remifentan
• the at least one sedative-hypnotic can be at least one selected from chloral hydrate, estazolam, flurazepam hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, temazepam, triazolam, zaleplon, zolpidem tartrate.
• the at least one anticonvulsant can be at least one selected from acetazolamide sodium, carbamazepine, clonazepam, clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde, fosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital, phenobarbital sodium, phenytoin, phenytoin sodium, phenytoin sodium (extended), primidone, tiagabine hydrochloride, topiramate, valproate sodium, valproic acid.
• the at least one antidepressant can be at least one selected from amitriptyline hydrochloride, amitriptyline pamoate, amoxapine, bupropion hydrochloride, citalopram hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin hydrochloride, fluoxetine hydrochloride, imipramine hydrochloride, imipramine pamoate, mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride, tranylcypromine sulfate, trimipramine maleate, venlafaxine hydrochloride.
• the at least one antianxiety drug can be at least one selected from alprazolam, buspirone hydrochloride, chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepate dipotassium, diazepam, doxepin hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam, mephrobamate, midazolam hydrochloride, oxazepam.
• the at least one antipsychotic drug can be at least one selected from chlo ⁇ romazine hydrochloride, clozapine, fluphenazine decanoate, fluephenazine enanthate, fluphenazine hydrochloride, haloperidol, haloperidol decanoate, haloperidol lactate, loxapine hydrochloride, loxapine succinate, mesoridazine besylate, molindone hydrochloride, olanzapine, pe ⁇ henazine, pimozide, prochlo ⁇ erazine, quetiapine fumarate, risperidone, thioridazine hydrochloride, thiothixene, thiothixene hydrochloride, trifluoperazine hydrochloride.
• the at least one central nervous system stimulant can be at least one selected from amphetamine sulfate, caffeine, dextroamphetamine sulfate, doxapram hydrochloride, methamphetamine hydrochloride, methylphenidate hydrochloride, modafinil, pemoline, phentermine hydrochloride.
• the at least one antiparkinsonian can be at least one selected from amantadine hydrochloride, benztropine mesylate, biperiden hydrochloride, biperiden lactate, bromocriptine mesylate, carbidopa-levodopa, entacapone, levodopa, pergolide mesylate, pramipexole dihydrochloride, ropinirole hydrochloride, selegiline hydrochloride, tolcapone, trihexyphenidyl hydrochloride.
• the at least one miscellaneous central nervous system drug can be at least one selected from bupropion hydrochloride, donepezil hydrochloride, droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine transdermal system, propofol, rizatriptan benzoate, sibutramine hydrochloride monohydrate, sumatriptan succinate, tacrine hydrochloride, zolmitriptan. (See, e.g., pp.
• the at least one cholinergic can be at least one selected from bethanechol chloride, edrophonium chloride, neostigmine bromide, neostigmine methylsulfate, physostigmine salicylate, pyridostigmine bromide.
• the at least one anticholinergics can be at least one selected from atropine sulfate, dicyclomine hydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate, propantheline bromide, scopolamine, scopolamine butylbromide, scopolamine hydrobromide.
• the at least one adrenergics can be at least one selected from atropine sulfate, dicyclomine hydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate, propantheline bromide, scopolamine, scopolamine butylbromide, scopolamine hydrobromide.
• the at least one adrenergic blocker can be at least one selected from dihydroergotamine mesylate, ergotamine tartrate, methysergide maleate, propranolol hydrochloride.
• the at least one skeletal muscle relaxant can be at least one selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol, tizanidine hydrochloride.
• the at least one neuromuscular blockers can be at least one selected from atracurium besylate, cisatracurium besylate, doxacurium chloride, mivacurium chloride, pancuronium bromide, pipecuronium bromide, rapacuronium bromide, rocuronium bromide, succinylcholine chloride, tubocurarine chloride, vecuronium bromide.
• the at least one antihistamine can be at least one selected from brompheniramine maleate, cetirizine hydrochloride, chlo ⁇ heniramine maleate, clemastine fumarate, cyproheptadine hydrochloride, diphenhydramme hydrochloride, fexofenadine hydrochloride, loratadine, promethazine hydrochloride, promethazine theoclate, triprolidine hydrochloride.
• the at least one bronchodilators can be at least one selected from albuterol, albuterol sulfate, aminophylline, atropine sulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate, epinephrine hydrochloride, ipratropium bromide, isoproterenol, isoproterenol hydrochloride, isoproterenol sulfate, levalbuterol hydrochloride, metaproterenol sulfate, oxtriphyllirie, pirbuterol acetate, salmeterol xinafoate, terbutaline sulfate, theophylline.
• the at least one expectorants or antitussives can be at least one selected from benzonatate, codeine phosphate, codeine sulfate, dextrametho ⁇ han hydrobromide, diphenhydramme hydrochloride, guaifenesin, hydromo ⁇ hone hydrochloride.
• the at least one miscellaneous respiratory drug can be at least one selected from acetylcysteine, beclomethasone dipropionate, beractant, budesonide, calfactant, cromolyn sodium, dornase alfa, epoprostenol sodium, flunisolide, fluticasone propionate, montelukast sodium, nedocromil sodium, palivizumab, triamcinolone acetonide, zafirlukast, zileuton. (See, e.g., pp.
• the at least one antacid, adsorbents, or antiflatulents can be at least one selected from aluminum carbonate, aluminum hydroxide, calcium carbonate, magaldrate, magnesium hydroxide, magnesium oxide, simethicone, sodium bicarbonate.
• the at least one digestive enymes or gallstone solubilizers can be at least one selected from pancreatin, pancrelipase, ursodiol.
• the at least one antidiarrheal can be at least one selected from attapulgite, bismuth subsalicylate, calcium polycarbophil, diphenoxylate hydrochloride or atropine sulfate, loperamide, octreotide acetate, opium tincture, opium tincure (camphorated).
• the at least one laxative can be at least one selected from bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagrada aromatic fluidextract, cascara sagrada fluidextract, castor oil, docusate calcium, docusate sodium, glycerin, lactulose, magnesium citrate, magnesium hydroxide, magnesium sulfate, methylcellulose, mineral oil, polyethylene glycol or electrolyte solution, psyllium, senna, sodium phosphates.
• the at least one antiemetic can be at least one selected from chlo ⁇ romazine hydrochloride, dimenhydrinate, dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizine hydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride, pe ⁇ henazine, prochlo ⁇ erazine, prochlo ⁇ erazine edisylate, prochlo ⁇ erazine maleate, promethazine hydrochloride, scopolamine, thiethylperazine maleate, trimethobenzamide hydrochloride.
• the at least one antiulcer drug can be at least one selected from cimetidine, cimetidine hydrochloride, famotidine, lansoprazole, misoprostol, nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate, ranitidine hydrochloride, sucralfate. (See, e.g., pp.
• the at least one coricosteroids can be at least one selected from betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate.
• the at least one androgen or anabolic steroids can be at least one selected from danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate, nandrolone phenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, testosterone transdermal system.
• the at least one estrogen or progestin can be at least one selected from esterified estrogens, estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal system, estradiol valerate, estrogens (conjugated), estropipate, ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradiol and norethindrone acetate, ethinyl estradiol and norgestimate, ethiny
• the at least one gonadroptropin can be at least one selected from ganirelix acetate, gonadoreline acetate, histrelin acetate, menotropins.
• the at least one antidiabetic or glucaon can be at least one selected from acarbose, chlo ⁇ ropamide, glimepiride, glipizide, glucagon, glyburide, insulins, metformin hydrochloride, miglitol, ( pioglitazone hydrochloride, repaglinide, rosiglitazone maleate, troglitazone.
• the at least one thyroid hormone can be at least one selected from levothyroxine sodium, liothyronine sodium, liotrix, thyroid.
• the at least one thyroid hormone antagonist can be at least one selected from methimazole, potassium iodide, potassium iodide (saturated solution), propylthiouracil, radioactive iodine (sodium iodide 131 I ), strong iodine solution.
• the at least one pituitary hormone can be at least one selected from corticotropin, cosyntropin, desmophressin acetate, leuprolide acetate, repository corticotropin, somatrem, somatropin, vasopressin.
• the at least one parathyroid-like drug can be at least one selected from calcifediol, calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol, etidronate disodium. (See, e.g., pp.
• the at least one diuretic can be at least one selected from acetazolamide, acetazolamide sodium, amiloride hydrochloride, bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid, furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone, spironolactone, torsemide, triamterene, urea.
• the at least one electrolyte or replacement solution can be at least one selected from calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic), calcium phosphate (tribasic), dextran (high-molecular-weight), dextran (low-molecular-weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, Ringer's injection, Ringer's injection (lactated), sodium chloride.
• the at least one acidifier or alkalinizer can be at least one selected from sodium bicarbonate, sodium lactate, tromethamine.
• the at least one hematinic can be at least one selected from ferrous fumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried), iron dextran, iron sorbitol, polysaccharide- iron complex, sodium ferric gluconate complex.
• the at least one anticoagulant can be at least one selected from ardeparin sodium, dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin calcium, heparin sodium, warfarin sodium.
• the at least one blood derivative can be at least one selected from albumin 5%, albumin 25%, antihemophilic factor, anti- inhibitor coagulant complex, antithrombin III (human), factor IX (human), factor IX complex, plasma protein fractions.
• the at least one thrombolytic enzyme can be at least one selected from alteplase, anistreplase, reteplase (recombinant), streptokinase, urokinase. (See, e.g., pp.
• the at least one alkylating drug can be at least one selected from busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride, melphalan, melphalan hydrochloride, streptozocin, temozolomide, thiotepa.
• the at least one antimetabolite can be at least one selected from capecitabine, cladribine, cytarabine, floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate, methotrexate sodium, thioguanine.
• the at least one antibiotic antineoplastic can be at least one selected from bleomycin sulfate, dactinomycin, daunorubicin citrate liposomal, daunorubicin hydrochloride, doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, epirubicin hydrochloride, idarabicin hydrochloride, mitomycin, pentostatin, plicamycin, valrubicin.
• the at least one antineoplastics that alter hormone balance can be at least one selected from anastrozole, bicalutamide, estramustine phosphate sodium, exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate, megestrol acetate, nilutamide, tamoxifen citrate, testolactone, toremifene citrate.
• the at least one miscellaneous antineoplastic can be at least one selected from asparaginase, bacillus
• Calmette-Guerin (live intravesical), dacarbazine, docetaxel, etoposide, etoposide phosphate, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimer sodium, procarbazine hydrochloride, rituximab, teniposide, topotecan hydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristine sulfate, vinorelbine tartrate. (See, e.g., pp.
• the at least one immunosuppressant can be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immune globulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetil hydrochloride. sirolimus, tacrolimus.
• the at least one vaccine or toxoid can be at least one selected from BCG vaccine, cholera vaccine, diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanus toxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanus toxoids and whole-cell pertussis vaccine, Haemophilius b conjugate vaccines, hepatitis A vaccine (inactivated), hepatisis B vaccine (recombinant), influenza virus vaccine 1999-2000 trivalent types A & B (purified surface antigen), influenza virus vaccine 1999-2000 trivalent types A & B (subvirion or purified subvirion), influenza virus vaccine 1999-2000 trivalent types A & B (whole virion), Japanese encephalitis virus vaccine (inactivated), Lyme disease vaccine (recombinant OspA), measles and mumps and rubella virus vaccine (live), measles and mumps and rubella virus
• the at least one antitoxin or antivenin can be at least one selected from black widow spider antivenin, Crotalidae antivenom (polyvalent), diphtheria antitoxin (equine), Micrurus fulvius antivenin).
• the at least one immune serum can be at least one selected from cytomegalovirus immune globulin (intraveneous), hepatitis B immune globulin (human), immune globulin intramuscular, immune globulin intravenous, rabies immune globulin (human), respiratory syncytial virus immune globulin intravenous (human), Rho(D) immune globulin (human), Rh 0 (D) immune globulin intravenous (human), tetanus immune globulin (human), varicella-zoster immune globulin.
• cytomegalovirus immune globulin intraveneous
• hepatitis B immune globulin human
• immune globulin intramuscular immune globulin intravenous
• rabies immune globulin human
• respiratory syncytial virus immune globulin intravenous human
• Rho(D) immune globulin human
• the at least one biological response modifiers can be at least one selected from aldesleukin, epoetin alfa, filgrastim, glatiramer acetate for injection, interferon alfacon-1, interferon alfa-2a (recombinant), interferon alfa-2b
• interferon gamma-lb levamisole hydrochloride
• oprelvekin sargramostim.
• the at least one ophthalmic anti-infectives can be selected form bacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin, gentamicin sulfate, ofloxacin 0.3%), polymyxin B sulfate, sulfacetamide sodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%>, tobramycin, vidarabine.
• the at least one ophthalmic anti- inflammatories can be at least one selected from dexamethasone, dexamethasone sodium phosphate, diclofenac sodium 0.1%, fluorometholone, flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate (suspension) prednisolone sodium phosphate (solution).
• the at least one miotic can be at least one selected from acetylocholine chloride, carbachol (intraocular), carbachol (topical), echothiophate iodide, piloca ⁇ ine, piloca ⁇ ine hydrochloride, piloca ⁇ ine nitrate.
• the at least one mydriatic can be at least one selected from atropine sulfate, cyclopentolate hydrochloride, epinephrine hydrochloride, epinephryl borate, homatropine hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide, tropicamide.
• the at least one ophthalmic vasoconstrictors can be at least one selected from naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride.
• the at least one miscellaneous ophthalmics can be at least one selected from apraclonidine hydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride, dorzolamide hydrochloride, emedastine difumarate, fluorescein sodium, ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranolol hydrochloride, sodium chloride (hypertonic), timolol maleate.
• the at least one otic can be at least one selected from boric acid, carbamide peroxide, chloramphenicol, triethanolamine polypeptide oleate-condensate.
• the at least one nasal drug can be at least one selected from beclomethasone dipropionate, budesonide, ephedrine sulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate, naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide, xylometazoline hydrochloride. (See, e.g., pp.
• the at least one local anti-infectives can be at least one selected from acyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazole nitrate, clindamycin phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate, metronidazole (topical), miconazole nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafine hydrochloride, terconazole, tetracycline hydrochloride, tioconazole, tolnaftate.
• the at least one scabicide or pediculicide can be at least one selected from crotamiton, lindane, permethrin, pyrethrins.
• the at least one topical corticosteroid can be at least one selected from betamethasone dipropionate, betamethasone valerate, clobetasol propionate, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate, fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasone propionate, halcionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate, triamcinolone acetonide.
• the at least one vitamin or mineral can be at least one selected from vitamin A, vitamin B complex, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol, paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodium fluoride, sodium fluoride (topical), trace elements, chromium, copper, iodine, manganese, selenium, zinc.
• the at least one calorics can be at least one selected from amino acid infusions (crystalline), amino acid infusions in dextrose, amino acid infusions with electrolytes, amino acid infusions with electrolytes in dextrose, amino acid infusions for hepatic failure, amino acid infusions for high metabolic stress, amino acid infusions for renal failure, dextrose, fat emulsions, medium-chain triglycerides. (See, e.g., pp.
• Hinge core mimetibody antibody or polypeptide compositions of the present invention can further comprise at least one of any suitable and/or effective amount of a composition or pharmaceutical composition comprising at least one hinge core mimetibody protein or antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF chemical or protein antagonist, TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptides thereof, or a small molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP- II), nerelimonmab, infliximab, enteracept, CDP-571, CDP-870, afelimomab, lenercept, and the like), an TNF antagonist (e.g., T
• Non- limiting examples of such cytokines include, but are not limted to, any of IL-1 to IL-23.
• Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely inco ⁇ orated herein by reference.
• Such compositions can also include toxin molecules that are associated, bound, co- formulated or co-administered with at least one antibody or polypeptide ofthe present invention.
• the toxin can optionally act to selectively kill the pathologic cell or tissue.
• the pathologic cell can be a cancer or other cell.
• Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, diphtheria toxin, a venom toxin, or a bacterial toxin.
• the term toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or viruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death.
• Such toxins may include, but are not limited to, enterotoxigenic E.
• coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like.
• Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E.
• coli e.g., strains of serotype 0157:H7
• Staphylococcus species e.g., Staphylococcus aureus, Staphylococcus pyogenes
• Shigella species e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei
• Salmonella species e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis
• Clostridium species e.g., Clostridium perfringens, Clostridium di ⁇ cile, Clostridium botulinum
• Camphlobacter species e.g., Camphlobacter jejuni
• Camphlobacter fetus Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonas aeruginosa, and Streptococci.
• Heliobacter species e.g., Heliobacter pylori
• Aeromonas species e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae
• Pleisomonas shigelloides Yersina enterocolitica
• Vibrios species e.g., Vibrios cholerae, Vibrios parahemolyticus
• hinge core mimetibody or specified portion or variant compositions ofthe present invention can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
• suitable auxiliaries are preferred.
• Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington 's Pharmaceutical Sciences, 18 U1 Edition, Mack Publishing Co. (Easton, PA) 1990.
• Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability ofthe hinge core mimetibody composition as well known in the art or as described herein.
• compositions include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
• Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
• Representative amino acid/hinge core mimetibody or specified portion or variant components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
• One preferred amino acid is glycine.
• Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
• monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
• disaccharides such as lactose, sucrose, trehalose,
• Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.
• hinge core mimetibody compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
• Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
• Preferred buffers for use in the present compositions are organic acid salts such as citrate.
• the hinge core mimetibody or specified portion or variant compositions ofthe invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ - cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
• polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ - cyclodext
• compositions according to the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19 th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference", 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely inco ⁇ orated herein by reference.
• Preferrred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.
• the invention provides for stable formulations, which can preferably include a suitable buffer with saline or a chosen salt, as well as optional preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one hinge core mimetibody or specified portion or variant in a pharmaceutically acceptable formulation.
• Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent.
• Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, O.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein.
• Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.
• 0.1-2% m-cresol e.g., 0.2, 0.3. 0.4, 0.5, 0.9
• the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one hinge core mimetibody or specified portion or variant with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
• the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one hinge core mimetibody or specified portion or variant, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one hinge core mimetibody or specified portion or variant in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
• the at least one hinge core mimetibody or specified portion or variant used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.
• the range of amounts of at least one hinge core mimetibody or specified portion or variant in the product ofthe present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ⁇ g/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
• the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
• preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.
• concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
• Other excipients e.g.
• isotonicity agents can be optionally and preferably added to the diluent.
• An isotonicity agent such as glycerin, is commonly used at known concentrations.
• a physiologically tolerated buffer is preferably added to provide improved pH control.
• the formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0.
• the formulations of the present invention have pH between about 6.8 and about 7.8.
• Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
• additives such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non- ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation.
• solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F
• the formulations ofthe present invention can be prepared by a process which comprises mixing at least one hinge core mimetibody or specified portion or variant and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent.
• a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof
• aqueous diluent Mixing the at least one hinge core mimetibody or specified portion or variant and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures.
• a suitable formulation for example, a measured amount of at least one hinge core mimetibody or specified portion or variant in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that may be optimized for the concentration and means of administration used.
• the claimed formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one hinge core mimetibody or specified portion or variant that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
• a preservative and/or excipients preferably a phosphate buffer and/or saline and a chosen salt
• Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus can provide a more convenient treatment regimen than currently available.
• the present claimed articles of manufacture are useful for administration over a period of immediately to twenty-four hours or greater.
• Formulations ofthe invention can optionally be safely stored at temperatures of from about 2 to about 40°C and retain the biologically activity ofthe protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to at least one of 1-12 months, one-half, one and a half, and/or two years.
• the solutions of at least one hinge core mimetibody or specified portion or variant in the invention can be prepared by a process that comprises mixing at least one hinge core mimetibody or specified portion or variant in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one hinge core mimetibody or specified portion or variant in water or buffer is combined in quantities sufficient to provide the protein and optionally a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that may be optimized for the concentration and means of administration used.
• the claimed products can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one hinge core mimetibody or specified portion or variant that is reconstituted with a second vial containing the aqueous diluent.
• a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
• the claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one hinge core mimetibody or specified portion or variant that is reconstituted with a second vial containing the aqueous diluent.
• the clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions ofthe at least one hinge core mimetibody or specified portion or variant solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients.
• Recognized devices comprising these single vial systems include those pen- injector devices for delivery of a solution such as Humaject ® 'NovoPen ® , B-D ® Pen, AutoPen ® , and OptiPen ® .
• Recognized devices comprising a dual vial system include those pen-injector systems for reconstituting a lyophilized drug in a cartridge for delivery ofthe reconstituted solution such as the HumatroPen ® .
• the products presently claimed include packaging material.
• the packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.
• the packaging material ofthe present invention provides instructions to the patient to reconstitute the at least one hinge core mimetibody or specified portion or variant in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product.
• the label indicates that such solution can be used over a period of 2-24 hours or greater.
• the presently claimed products are useful for human pharmaceutical product use.
• the formulations ofthe present invention can be prepared by a process that comprises mixing at least one hinge core mimetibody or specified portion or variant and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt.
• aqueous diluent Mixing the at least one hinge core mimetibody or specified portion or variant and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures.
• a suitable formulation for example, a measured amount of at least one hinge core mimetibody or specified portion or variant in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
• the claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one hinge core mimetibody or specified portion or variant that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent.
• a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
• At least one hinge core mimetibody or specified portion or variant in either the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or EVI injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
• the present invention for mimetibodies also provides a method for modulating or treating anemia, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of any anemia, cancer treatment related anemia, radiotherapy or chemotherapy related anemia, viral or bacterial infection treatment related anemia, renal anemia, anemia of prematurity, pediatric and/or adult cancer-associated anemia, anemia associated with lymphoma, myeloma, multple myeloma, AIDS-associated anemia, concomitant treatment for patients with or without autologous blood donation awaiting elective surgery, preoperatve and post operative for surgery, autologous blood donation or transfusion, perioperative management, cyclic neutropenia or Kostmann syndrome (congenital agranulocytosis), end- stage renal disease, anemia associated with dialysis, chronic renal insufficiency, primary hemopoietic diseases, such as congenital hypoplastic anemia, thalassemia major, or sickle cell disease
• Mimetibodies of the present invention can also be used for non-renal forms of anemia induced, for example, by chronic infections, inflammatory processes, radiation therapy, and cytostatic drug treatment, and encouraging results in patients with non-renal anemia have been reported.
• the present invention also provides a method for modulating or treating an anemia or blood cell related condition, in a cell, tissue, organ, animal, or patient, wherein said anemia or blood cell related condition is associated with at least one including, but not limited to, at least one of immune related disease, cardiovascular disease, infectious, malignant and/or neurologic disease.
• Such a method can optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• the present invention also provides a method for modulating or treating cancer/infecteous disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia , epiglottitis, e.
• coli 0157:h7 hemolytic uremic syndrome/thrombolytic thrombocytopenic pu ⁇ ura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellulare, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epidydimitis, legionella, lyme disease, influenza a, epstein-barr virus, vital-associated hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and the like; (ii) leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL),
• Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• Such a method can optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• the present invention also provides a method for modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, diabetic ateriosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis ofthe cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrytrrmias, ventricular fibrillation, His bundle arrythmias, atrioventricular block, bundle branch block, myocardial ischemic disorders, coronary
• Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• Any method ofthe present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• Such a method can optionally further comprise co- administration or combination therapy for treating such immune diseases, wherein the administering of said at least one hinge core mimetibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a sulfonamide,
• Mimetibodies can also be used ex vivo, such as in autologous marrow culture. Briefly, bone marrow is removed from a patient prior to chemotherapy and treated with TPO and/or EPO, optionally in combination with mimetibodies, optionally in combination with one or more additional cytokines. The treated marrow is then returned to the patient after chemotherapy to speed the recovery ofthe marrow.
• TPO can also be used for the ex vivo expansion of marrow or peripheral blood progenitor (PBPC) cells.
• PBPC peripheral blood progenitor
• marrow Prior to chemotherapy treatment, marrow can be stimulated with stem cell factor (SCF) or G-CSF to release early progenitor cells into peripheral circulation.
• SCF stem cell factor
• G-CSF G-CSF
• progenitors are optionally collected and concentrated from peripheral blood and then treated in culture with TPO and mimetibodies, optionally in combination with one or more other cytokines, including but not limited to SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11, to differentiate and proliferate into high-density megakaryocyte cultures, which are optionally then be returned to the patient following high-dose chemotherapy.
• cytokines including but not limited to SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL-11.
• Doses of TPO for ex vivo treatment of bone marrow will be in the range of 100 pg/ml to 10 ng/ml, preferably 500 pg/ml to 3 ng/ml.
• TNF antagonists suitable for compositions, combination therapy, co-administration, devices and/or methods ofthe present invention include, but are not limited to, anti-TNF antibodies, ligand-binding fragments thereof, and receptor molecules which bind specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signalling, such as mitogen activated protein (MAP)
• MAP mitogen activated protein
• a "tumor necrosis factor antibody,” “TNF antibody,” “TNF ⁇ antibody,” or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNF ⁇ activity in vitro, in situ and/or preferably in vivo.
• a suitable TNF human antibody ofthe present invention can bind TNF ⁇ and includes anti-TNF antibodies, antigen- binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNF ⁇ .
• a suitable TNF antibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
• Chimeric antibody cA2 consists ofthe antigen binding variable region ofthe high- affinity neutralizing mouse anti-human TNF ⁇ IgGl antibody, designated A2, and the constant regions of a human IgGl, kappa immunoglobulin.
• the human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half-life and decreases the immunogenicity of the antibody.
• the avidity and epitope specificity ofthe chimeric antibody cA2 is derived from the variable region of the murine antibody A2.
• a preferred source for nucleic acids encoding the variable region ofthe murine antibody A2 is the A2 hybridoma cell line.
• Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant human TNF ⁇ in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNF ⁇ , the affinity constant of chimeric antibody cA2 was calculated to be 1.04xl0 10 M " '. Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow, et ah, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et ah, eds., Current Protocols in Immunology, Greene Publishing Assoc.
• murine monoclonal antibody A2 is produced by a cell line designated cl34A.
• Chimeric antibody cA2 is produced by a cell line designated cl68A. Additional examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (see, e.g., U.S. Patent No. 5,231,024; M ⁇ ller, A. et al, Cytokine 2(3): 162-169 (1990); U.S. Application No.
• TNF Receptor Molecules Preferred TNF receptor molecules useful in the present invention are those that bind TNF ⁇ with high affinity (see, e.g., Feldmann et al, International Publication No. WO 92/07076 (published April 30, 1992); Schall et al, Cell 61:361-310 (1990); and Loerscher et al, Cell (57:351-359 (1990), which references are entirely inco ⁇ orated herein by reference) and optionally possess low immunogenicity.
• the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention.
• Truncated forms of these receptors comprising the extracellular domains (ECD) ofthe receptors or functional portions thereof (see, e.g., Corcoran et al, Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present invention.
• Truncated forms ofthe TNF receptors, comprising the ECD have been detected in urine and serum as 30 kDa and 40 kDa TNF ⁇ inhibitory binding proteins (Engelmann, H. et al, J. Biol. Chem. 2(55:1531-1536 (1990)).
• TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules which are useful in the methods and compositions of the present invention.
• the TNF receptor molecules which can be used in the invention are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved.
• TNF receptor multimeric molecules useful in the present invention comprise all or a functional portion ofthe ECD of two or more TNF receptors linked via one or more polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG).
• the multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression ofthe multimeric molecule.
• TNF immunoreceptor fusion molecules useful in the methods and compositions ofthe present invention comprise at least one portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These in ⁇ m ⁇ norecepto r fusion molecules can be assembled as monomers, or hetero- or homo-multimers.
• the immunoreceptor fusion molecules can also be monovalent or multivalent.
• TNF immunoreceptor fusion molecules TNF receptor/IgG fusion protein.
• TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al, Eur. J. Immunol 27:2883-2886 (1991); Ashkenazi et ah, Proc. Natl. Acad. Sci. USA #5:10535-10539 (1991); Peppel et ah, J. Exp. Med. 774:1483-1489 (1991); Kolls et ah, Proc. Natl. Acad. Sci.
• a functional equivalent, derivative, fragment or region of TNF receptor molecule refers to the portion ofthe TNF receptor molecule, or the portion ofthe TNF receptor molecule sequence which encodes TNF receptor molecule, that is of sufficient size and sequences to functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNF ⁇ with high affinity and possess low immunogenicity).
• a functional equivalent of TNF receptor molecule also includes modified TNF receptor molecules that functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNF ⁇ with high affinity and possess low immunogenicity).
• a functional equivalent of TNF receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et al,
• Cytokines include, but are not limited to all known cytokines. See, e.g., CopewithCytolcines.com.
• Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof. Any method ofthe present invention can comprise a method for treating a protein mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one hinge core mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
• Such a method can optionally further comprise co-administration or combination therapy for treating such immune diseases, wherein the administering of said at least one hinge core mimetibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one other cytokines such as IL-3, IL -6 and IL -11; stem cell factor; G-CSF and GM-CSF.
• cytokines such as IL-3, IL -6 and IL -11
• stem cell factor such as IL-3, IL -6 and IL -11
• G-CSF stem cell factor
• GM-CSF GM-CSF
• treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one hinge core mimetibody composition that total, on average, a range from at least about 0.01 to 500 milligrams of at least one hinge core mimetibody or specified portion or variant /kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams hinge core mimetibody or specified portion or variant /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
• the effective serum concentration can comprise 0.1-5000 ⁇ g/ml serum concentration per single or multiple adminstration.
• Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity ofthe composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
• Preferred doses can optionally include 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 009, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 mg/kg/administration, or any range, value or fraction thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0,
• the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight ofthe recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
• a dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
• 0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
• treatment of humans or animals can be provided as a onetime or periodic dosage of at least one hinge core mimetibody or specified portion or variant ofthe present invention 0.01 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single, infusion or repeated doses.
• Dosage forms (composition) suitable for internal administration generally contain from about 0.0001 milligram to about 500 milligrams of active ingredient per unit or container.
• the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight ofthe composition.
• the hinge core mimetibody or specified portion or variant can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
• the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
• the formulation is sterilized by known or suitable techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
• Therapeutic Administration Many known and developed modes of can be used according to the present invention for administering pharmaceutically effective amounts of at least one hinge core mimetibody or specified portion or variant according to the present invention. While pulmonary administration is used in the following description, other modes of administration can be used according to the present invention with suitable results.
• a hinge core mimetibody ofthe present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
• Parenteral Formulations and Administration Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
• Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.
• Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aquous solution or a sterile injectable solution or suspension in a solvent.
• As the usable vehicle or solvent water, Ringer's solution, isotonic saline, etc.
• sterile involatile oil can be used as an ordinary solvent, or suspending solvent.
• any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri- glycerides.
• Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely inco ⁇ orated herein by reference.
• the invention further relates to the administration of at least one hinge core mimetibody or specified portion or variant by parenteral, subcutaneous, intramuscular, intravenous, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.
• Protein, hinge core mimetibody or specified portion or variant compositions can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for buccal, or sublingual administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols or certain agents; or transdermally particularly in the form of a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al.
• parenteral subcutaneous, intramuscular or intravenous
• vaginal or rectal administration particularly in semisolid forms such as creams and suppositories
• buccal, or sublingual administration particularly in the form of tablets or capsules
• At least one hinge core mimetibody or specified portion or variant composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
• at least one hinge core mimetibody or specified portion or variant can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of hinge core mimetibody or specified portion or variants are also known in the art.
• Nebulizers like AERxTM Aradigm, the Ultravent ® nebulizer (Mallinckrodt), and the Acorn II ® nebulizer (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above references entirely inco ⁇ orated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols.
• These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.
• a composition comprising at least one hinge core mimetibody or specified portion or variant is delivered by a dry powder inhaler or a sprayer.
• an inhalation device for administering at least one hinge core mimetibody or specified portion or variant ofthe present invention.
• delivery by the inhalation device is advantageously reliable, reproducible, and accurate.
• the inhalation device can optionally deliver small dry particles, e.g. less than about 10 ⁇ m, preferably about 1-5 ⁇ m, for good respirability.
• hinge core mimetibody or specified portion or variant Compositions as a Spray
• a spray including hinge core mimetibody or specified portion or variant composition protein can be produced by forcing a suspension or solution of at least one hinge core mimetibody or specified portion or variant through a nozzle under pressure.
• the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
• An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
• particles of at least one hinge core mimetibody or specified portion or variant composition protein delivered by a sprayer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
• Formulations of at least one hinge core mimetibody or specified portion or variant composition protein suitable for use with a sprayer typically include hinge core mimetibody or specified portion or variant composition protein in an aqueous solution at a concentration of about 1 mg to about 20 mg of at least one hinge core mimetibody or specified portion or variant composition protein per ml of solution.
• the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
• the formulation can also include an excipient or agent for stabilization ofthe hinge core mimetibody or specified portion or variant composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
• Bulk proteins useful in formulating hinge core mimetibody or specified portion or variant composition proteins include albumin, protamine, or the like.
• Typical carbohydrates useful in formulating hinge core mimetibody or specified portion or variant composition proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like.
• the hinge core mimetibody or specified portion or variant composition protein formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation ofthe hinge core mimetibody or specified portion or variant composition protein caused by atomization ofthe solution in forming an aerosol.
• a surfactant which can reduce or prevent surface-induced aggregation ofthe hinge core mimetibody or specified portion or variant composition protein caused by atomization ofthe solution in forming an aerosol.
• Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14%) by weight of the formulation.
• Especially preferred surfactants for pu ⁇ oses of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as mimetibodies, or specified portions or variants, can also be included in the formulation.
• Nebulizer hinge core mimetibody or specified portion or variant composition protein can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
• a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
• a compressed air source is used to create a high-velocity air jet through an orifice.
• a low-pressure region is created, which draws a solution of hinge core mimetibody or specified portion or variant composition protein through a capillary tube connected to a liquid reservoir.
• the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
• a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
• high- frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the formulation of hinge core mimetibody or specified portion or variant composition protein either directly or through a coupling fluid, creating an aerosol including the hinge core mimetibody or specified portion or variant composition protein.
• particles of hinge core mimetibody or specified portion or variant composition protein delivered by a nebulizer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
• Formulations of at least one hinge core mimetibody or specified portion or variant suitable for use with a nebulizer, either jet or ultrasonic typically include hinge core mimetibody or specified portion or variant composition protein in an aqueous solution at a concentration of about 1 mg to about 20 mg of at least one hinge core mimetibody or specified portion or variant protein per ml of solution.
• the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
• the formulation can also include an excipient or agent for stabilization ofthe at least one hinge core mimetibody or specified portion or variant composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
• Bulk proteins useful in formulating at least one hinge core mimetibody or specified portion or variant composition proteins include albumin, protamine, or the like.
• Typical carbohydrates useful in formulating at least one hinge core mimetibody or specified portion or variant include sucrose, mannitol, lactose, trehalose, glucose, or the like.
• the at least one hinge core mimetibody or specified portion or variant formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation ofthe at least one hinge core mimetibody or specified portion or variant caused by atomization ofthe solution in forming an aerosol.
• a surfactant which can reduce or prevent surface-induced aggregation ofthe at least one hinge core mimetibody or specified portion or variant caused by atomization ofthe solution in forming an aerosol.
• Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between 0.001 and 4% by weight ofthe formulation.
• Especially preferred surfactants for pu ⁇ oses of this invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as hinge core mimetibody or specified portion or variant protein can also be included in the formulation.
• a propellant in a metered dose inhaler (MDI)
• a propellant at least one hinge core mimetibody or specified portion or variant, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas.
• Actuation ofthe metering valve releases the mixture as an aerosol, preferably containing particles in the size range of less than about 10 ⁇ m, preferably about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
• the desired aerosol particle size can be obtained by employing a formulation of hinge core mimetibody or specified portion or variant composition protein produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like.
• Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
• Formulations of at least one hinge core mimetibody or specified portion or variant for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one hinge core mimetibody or specified portion or variant as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
• the propellant can be any conventional material employed for this pu ⁇ ose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane- 227), or the like.
• the propellant is a hydrofluorocarbon.
• the surfactant can be chosen to stabilize the at least one hinge core mimetibody or specified portion or variant as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
• Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as protein can also be included in the formulation.
• One of ordinary skill in the art will recognize that the methods of the current invention can be achieved by pulmonary administration of at least one hinge core mimetibody or specified portion or variant compositions via devices not described herein.
• compositions and methods of administering at least one hinge core mimetibody or specified portion or variant include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes abso ⁇ tion through mucosal surfaces by achieving mucoadhesion ofthe emulsion particles (U.S. Pat. Nos. 5,514,670).
• Mucous surfaces suitable for application ofthe emulsions ofthe present invention can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration.
• Formulations for vaginal or rectal administration can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.
• Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops.
• excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. No. 5,849,695).
• Oral Formulations and Administration for oral rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation.
• adjuvants e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether
• enzymatic inhibitors e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol
• the active constituent compound ofthe solid- type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol . dextran. starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arable, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
• These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc. Tablets and pills can be further processed into enteric-coated preparations.
• the liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations allowable for medical use. These preparations may contain inactive diluting agents ordinarily used in said field, e.g., water.
• Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents orally are known in the art.
• the at least one hinge core mimetibody or specified portion or variant is encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
• a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
• suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. No. 5,814,599).
• a dosage form can contain a pharmaceutically acceptable non-toxic salt ofthe compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-dibenzy
• the compounds ofthe present invention or, preferably, a relatively insoluble salt such as those just described can be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
• Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
• Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non- antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919.
• the compounds or, preferably, relatively insoluble salts such as those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals.
• Additional slow release, depot or implant formulations, e.g. gas or liquid liposomes are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).
• a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the hinge core mimetibody or specified portion or variant coding sequence, and signals required for the termination of transcription and polyadenylation ofthe transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter ofthe cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
• LTRS long terminal repeats
• CMV cytomegalovirus
• Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
• vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hy
• Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
• the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
• the co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation ofthe transfected cells.
• the transfected gene can also be amplified to express large amounts ofthe encoded hinge core mimetibody or specified portion or variant.
• the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies ofthe gene of interest.
• Another useful selection marker is the enzyme glutamine synthase (GS) (Mu ⁇ hy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and
• NSO cells are often used for the production of hinge core mimetibody or specified portion or variants.
• the expression vectors pCl and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment ofthe CMV-enhancer (Boshart, et al, Cell 41:521-530 (1985)).
• LTR Rous Sarcoma Virus
• CMV-enhancer Boshart, et al, Cell 41:521-530 (1985)
• Multiple cloning sites e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning ofthe gene of interest.
• the vectors contain in addition the 3' intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.
• Cloning and Expression in CHO Cells The vector pC4 is used for the expression of hinge core mimetibody or specified portion or variant. Plasmid pC4 is a derivative ofthe plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouse DHFR gene under control ofthe SV40 early promoter.
• Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
• a selective medium e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD
• methotrexate methotrexate
• the amplification ofthe DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., F. W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A.
• DHFR target enzyme
• a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies ofthe amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated into one or more chromosome(s) ofthe host cell.
• Plasmid pC4 contains for expressing the gene of interest the strong promoter ofthe long terminal repeat (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438- 447 (1985)) plus a fragment isolated from the enhancer ofthe immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)). Downstream ofthe promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration ofthe genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site ofthe rat preproinsulin gene.
• LTR long terminal repeat
• CMV cytomegalovirus
• high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
• Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the EPO in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
• Other signals e.g., from the human growth hormone or globin genes can be used as well.
• Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
• the plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.
• the DNA sequence encoding the complete hinge core mimetibody or specified portion or variant is used, corresponding to HC and LC variable regions of a hinge core mimetibody of the present invention, according to known method steps.
• Isolated nucleic acid encoding a suitable human constant region (i.e., HC and LC regions) is also used in this construct.
• the isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase.
• E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
• Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection. 5 ⁇ g ofthe expression plasmid pC4 is cotransfected with 0.5 ⁇ g ofthe plasmid pSV2-neo using lipofectin.
• the plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
• the cells are seeded in alpha minus MEM supplemented with 1 ⁇ g /ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g /ml G418.
• single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100 - 200 mM. Expression ofthe desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
• Additional constructs can be expressed with single or multiple amino acid changes in order to avoid undesirable activities. These changes may be expressed alone or multiple changes combined in a single construct.
• the cysteine normally involved in a disulfide bridge between the HC and LC will be mutated to an alanine. While this cysteine may be involved in stabilizing the construct by forming a third disulfide bridge, it is possible that it may aberrantly form a disulfide bond with other cyseines within the construct, or it could form a disulfide linkage between two constructs. By removing the cysteine, proper folding and assembly could be enhanced and the possibility of self-association diminished.
• ADCC Alzheimer's disease .
• Similar changes can be made in the hinge region of other immunoglobulin classes and subclasses.
• Another modification that would result in a decrease in mediation of immune effector functions is the removal ofthe glycosylation attachment site. This can be accomplished by mutation ofthe asparagine to glutamine (Q).
• Aglycosylated versions ofthe IgGl subclass are known to be poor mediators of immune effector function (Jefferis et al. 1998, Immol. Rev., ..-! 163, 50-76).
• MHC binding epitopes within the mimetibody were analyzed. Mutations that would decrease the predicted immunogenicity of one or more peptides are evaluated for in vivo effect or. immunogenicity.
• the mimetibody constructs described above offers an alternative way of displaying bioactive peptides.
• proposed modifications are expected to, in combination and in addition to the novel features of mimetibodies ofthe present invention, enhance their utility. It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations ofthe present invention are possible in light of the above teachings and, therefore, are within the scope ofthe present invention (

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